Structure-property-relations of cuticular photonic crystals evolved by different beetle groups (Insecta, Coleoptera)

Von der Fakultät für Georessourcen und Materialtechnik der Rheinisch-Westfälischen Technischen Hochschule Aachen

zur Erlangung des akademischen Grades eines Doktors der Ingenieurwissenschaften

genehmigte Dissertation vorgelegt von M.Sc.

Xia Wu

aus Jiangsu, China

Berichter: Professor Dr.-Ing. Dierk Raabe Univ.-Prof. Dr.-Ing. Andreas Bührig-Polaczek

Tag der mündlichen Prüfung: 20. Januar 2014

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar

Imprint Copyright: © 2014 Xia Wu Printed and published by: epubli GmbH, Berlin, www.epubli.de ISBN 978-3-8442-9496-5 D 82 (Diss. RWTH Aachen University, 2014)

To my family 致我的家人

Acknowledgements

Acknowledgements

I wish to thank Prof. Dr. Dierk Raabe for giving me the opportunity to work on this really interesting topic and for providing the excellent working conditions as well as his encouragement, inspiring discussions and guidance in this study. I also wish to thank Prof. Dr. Andreas Bührig-Polaczek, who agreed to be the co-advisor of this thesis. I am very thankful to Dr. Helge Fabritius for his patient guidance, many inspiring discussions and kind help with the biological part and many other aspects of this work. Many thanks go to Dr. Andreas Erbe for many fruitful discussions and his guidance and help in both theoretical and experimental parts of optics and in chemistry as well. I am also thankful to the helps from Dr. Anna Janus, Jin Lu, Simone Karsten with whom I worked together in the Biological Composites group. I am very grateful to the fruitful collaborations in biotemplating and many inspiring discussions with Prof. Dr. Cordt Zollfrank, Dr. Daniel Van Opdenbosch and Maren Johannes. I would like to thank my colleagues Prof. Dr. Svetoslav Nikolov, Prof. Dr. Philip Eisenlohr, Dr. Duancheng Ma, Dr. Bin Liu, Dr. Lifang Zhu, Martin Diehl for the help and discussion on simulation works, and Katja Angenendt, Monika Nellessen and Petra Ebbinghaus for their help with the experiments. Many thanks also go to Dr. Yujiao Li, Dr. Lei Yuan, Dr. Ying Chen, Dr. Tao Liu, Dr. Jingbo Song, Dr. Nan Jia, Zongrui Pei and many other Chinese colleagues who have helped me in my study and personal life. I am very grateful to Dr. Joachim Holstein from the Entomology department of the Staatliches Museum für Naturkunde, Stuttgart who provided the beetle specimens used for this study. Finally, I take this opportunity to express my profound gratitude to my wife Zhe for her support in my personal life, and to my beloved parents and parents in law as well.

I

Table of Contents

Table of Contents

Acknowledgements ...... I Foreword ...... - 1 - Chapter 1. Introduction ...... - 3 - Chapter 2. Biological and Physical Background ...... - 7 - 2.1. Evolutionary origin of structural colors ...... - 7 - 2.2. Structure and chemical composition of insect cuticle ...... - 7 - 2.3. Physical background of photonic structures occurring in nature ...... - 15 - 2.3.1. Photonic crystals ...... - 15 - 2.3.2. Multilayer structures ...... - 16 - 2.3.3. Helicoidal photonic structures ...... - 18 - 2.3.4. 3D photonic crystals ...... - 20 - Chapter 3. Materials and Methods ...... - 25 - 3.1. Materials ...... - 25 - 3.2. Sample preparation ...... - 25 - 3.3. Structural characterization ...... - 26 - 3.4. Chemical characterization ...... - 27 - 3.5. Optical characterization ...... - 27 - 3.6. Optical simulation ...... - 29 - Chapter 4. Multilayer Structures (Ground Beetles) ...... - 31 - 4.1. Introduction ...... - 31 - 4.2. Structural characterization ...... - 32 - 4.2.1. Top surfaces of original samples ...... - 32 - 4.2.2. Cross-sectional surfaces of fractured samples ...... - 33 - 4.2.3. Cross-sectional surfaces of microtomed samples .... - 36 - 4.2.4. Oblique sections of microtomed samples ...... - 38 -

III

Table of Contents

4.2.5. Structure and color changes of NaOH treated samples ...... - 42 - 4.3. Experimental investigation of the optical properties ...... - 48 - 4.3.1. Reflectance at normal incidence ...... - 48 - 4.3.2. Reflectance at different angles of incidence ...... - 49 - 4.3.3. Scattering at different angles of incidence ...... - 50 - 4.4. Simulation of the optical properties ...... - 51 - 4.4.1. Reflectance at normal incidence ...... - 51 - 4.4.2. Influence of different angles of incidence...... - 53 - 4.4.3. Influence of the number of bi-layers ...... - 53 - 4.4.4. Influence of varying thicknesses of individual layers ...... - 54 - 4.4.5. Influence of the thickness of the outermost layer .... - 55 - 4.4.6. Influence of the thickness of the transparent surface layer ...... - 57 - 4.5. Discussion ...... - 58 - 4.5.1. Photonic structures of the Ground beetle species .... - 58 - 4.5.2. Optical properties of the photonic structures ...... - 64 - 4.6. Summary ...... - 70 - Chapter 5. Helicoidal Photonic Structures (Scarab Beetles) ..... - 73 - 5.1. Introduction ...... - 73 - 5.2. Structural characterization ...... - 75 - 5.2.1. Cross- and transverse-sectional surfaces of fractured samples ...... - 75 - 5.2.2. Section surfaces of microtomed samples ...... - 79 - 5.2.3. Structure changes of NaOH treated samples...... - 86 - 5.3. Optical characterization ...... - 88 - 5.3.1. Reflectance at normal incidence ...... - 88 - 5.3.2. Polarization effect ...... - 88 - 5.4. Discussion ...... - 89 - 5.4.1. Photonic structures of the Scarab beetle species ...... - 89 - 5.4.2. Optical properties of the photonic structures ...... - 92 - 5.5. Summary ...... - 93 -

IV

Table of Contents

Chapter 6. Three-dimensional Photonic Crystals (Weevils) ..... - 97 - 6.1. Introduction ...... - 97 - 6.2. Structural characterization ...... - 99 - 6.2.1. Intact and broken scales ...... - 99 - 6.2.2. Microtome polished scales ...... - 101 - 6.2.3. Focused ion beam (FIB) milled scales ...... - 104 - 6.3. Chemical characterization of the transparent domains . - 110 - 6.3.1. Energy-dispersive X-ray (EDX) spectroscopy ...... - 110 - 6.3.2. Fourier transform infrared (FTIR) spectroscopy ... - 111 - 6.4. Photonic band structure calculation ...... - 112 - 6.5. Optical characterization ...... - 115 - 6.5.1. Selective reflections of the scales ...... - 115 - 6.5.2. Polarization effect ...... - 117 - 6.6. Discussion ...... - 124 - 6.6.1. Structural origins of the scales ...... - 124 - 6.6.2. Photonic crystals in the core ...... - 125 - 6.6.3. Optical properties of the scales ...... - 128 - 6.6.4. Polarization effect ...... - 133 - 6.7. Summary ...... - 139 - Chapter 7. Conclusions ...... - 143 - Appendix ...... - 147 - Bibliography ...... - 149 - Abstract ...... - 161 - Zusammenfassung ...... - 163 -

V

Foreword

Foreword

Throughout human history, understanding and controlling the microstructure of materials has led to improvement and optimization of their properties. Major advances in technology followed. One emerging field in materials science is the design and fabrication of so- called photonic crystals, which are expected to play the same important role in photonics as semiconductors do in electronics in the near future. In photonic crystals, dielectric materials are periodically arranged in space, analogous to atoms or molecules at lattice points in “classical” crystals. The Bragg diffraction of light by photonic crystals generates frequency ranges acting as photonic band gaps. Light with frequencies within these gaps is reflected and cannot propagate through the photonic structure. Therefore, photonic crystals can be regarded as optical analogues to semiconductors possessing electronic band gaps. The theories of solid-state physics used to characterize and analyze electronic band structures have been transformed in the context of electromagnetics to be applied in the field of photonic band gap materials. Structural characteristics of classical crystals like defects, symmetries, crystallographic orientations that govern the mechanical and electrical properties of materials all found their counterparts in photonic crystals where they play important roles in controlling the optical properties. Structures that act as photonic crystals can have different degrees of complexity ranging from one dimensional to three dimensional depending on whether the refractive indices of the constituting phases vary in one, two or three directions. This spatial periodicity represents the lattice parameter of photonic crystals that determine the wavelength ranges of the reflected light. Today’s technology is still facing challenges in producing complex three-dimensional photonic crystals that operate in the spectral range of visible light due to limitations in the fabrication methods. In nature, however, many living organisms including plants, fish, birds and insects have evolved biological photonic crystals that can generate vivid and iridescent structural colors and have therefore attracted the

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Foreword attention of both biologists and material scientists. As most biological materials, these photonic crystals are composites formed using the limited set of constituents available to the organisms through their genetic predisposition. The lack of diversity in constituents has been compensated by the evolution of a large variety of different, highly sophisticated structures to achieve the required optical properties. In this study, I selected different beetle species to systematically investigate the microstructure of their photonic crystals and correlate it to the resulting optical properties with the goal to evaluate their different design principles. In addition, biological optically active structures are often part of organs or tissues that are not primarily formed for optical purposes, but have functions that require other physical properties. In beetles, the photonic crystals are formed by the cuticle, a tissue whose main function is to act as an exoskeleton and provide appropriate mechanical properties. Thus, such biological structures are promising sources of inspiration for the design and fabrication of innovative multifunctional composite materials with a balanced combination of optical properties and other physical properties, e.g. of mechanical nature, that can be used in modern technical applications.

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Chapter 1 | Introduction

Chapter 1. Introduction

The beauty and significance of structural colors generated by biological photonic structures have already very early attracted the attention of pioneer scientists, such as Hooke, Newton and Darwin, from different disciplines of natural science. With the help of the electromagnetic theory of light and high resolution investigative methods like electron microscopy, scientists are now able to characterize the microstructure of biological photonic structures down to the nanometer-scale and understand the mechanisms of how light interacts with these structures (Kinoshita, 2008; Doucet and Meadows, 2009). The increasing interest in the field of photonic band gap materials, which are the optical analogues to semiconductors (Soukoulis, 2001; Joannopoulos et al., 2008), is another reason that encouraged scientists to investigate and characterize their biological counterparts, the natural photonic crystals (Vukusic and Sambles, 2003; Kinoshita and Yoshioka, 2005b; Kinoshita, 2008). In nature, colors are produced by two major mechanisms: light- absorption by pigments and physical interaction of light with nanometer-scale (most are ~ 100 nm) photonic structures. In some cases, these two mechanisms are combined to generate specific color effects (Shawkey et al., 2009). The structural colors produced by biological photonic crystals can be brighter and more saturated than pigmentary colors when viewed at certain angles (Vukusic et al., 1999; Osorio and Ham, 2002; Doucet and Meadows, 2009). A classic example is the brilliant blue coloration of the wings of the Morpho rhetenor butterfly that are even visible from a distance of half a mile away (Vukusic and Sambles, 2003). While pigments usually degrade after the death of an organism, photonic crystals can be remarkably stable. For instance, the multilayer photonic structures in the cuticle of fossil beetles (Parker and McKenzie, 2003; McNamara et al., 2011a) and scales of moths (McNamara et al., 2011b) that lived 47 million years ago were preserved well enough to display their structural colors even today. The same has also been observed for fossil feathers

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Introduction

(Clarke et al., 2010; Vinther et al., 2010). In addition to their optical function, biological photonic crystals are mostly formed as parts of specific biological tissues or materials and that have to perform additional functions simultaneously. Thus, they can frequently be regarded as multifunctional materials. For example, the photonic crystals in scales covering the wings of butterflies should not only provide colors, but also be light-weight at the same time, which is crucial for the ability to fly. Such examples show that biological photonic crystals are interesting model systems for materials scientists since they combine attractive optical properties, stability and often multiple functionalities in one composite material. In the animal kingdom, particularly insects are renowned for their ability to display vivid and diverse color effects. Among them, butterflies (Lepidoptera) and beetles (Coleoptera) have evolved a large range of biological photonic structures and thus attract the most attention from biologists, physicists and materials scientists. Investigation of the structure-property relations of biological photonic structures and thus understanding their design principles has already inspired various applications. For instance, the multilayer structure in the scales of Morpho butterflies (Tabata et al., 1996) led to the development of synthetic structurally colored polymer fibers and chips that are used in the textile industry and as paint additives in the automobile industry (Iohara et al., 2000). The same structure was demonstrated to be a suitable design for new synthetic optical gas sensors (Potyrailo et al., 2007). The “sandwich”-type photonic structures like the helicoidal-unidirectional-helicoidal structures occurring in the exocuticle of the beetle Plusiotis resplendens (Caveney, 1971) inspired the design of an electro-tunable optical diode (Hwang et al., 2005). The random arrangement and optimized packing density of cuticular filaments inside the ultrathin (~5 µm) white scales of the beetle Cyphochilus spp. (Vukusic et al., 2007) were technically transferred into a more efficient bright and white mineral coating (Hallam et al., 2009). These examples demonstrate the high potential of photonic crystals formed by insects for biomimetic optical engineering, which is the major motivation for this study.

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Chapter 1 | Introduction

I chose three different groups of beetles (Carabidae, Scarabeidae and Curculionidae) which evolved distinct biological photonic structures located in different regions of their exoskeleton as examples to evaluate their design principles and explore their potential for technological applications. The creativity and flexibility of these three representative groups of beetles to generate different optical effects by both structural and chemical modifications of the same basic material (cuticle) is the reason for selecting them as showcases.

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Chapter 2 | Biological and Physical Background

Chapter 2. Biological and Physical Background

2.1. Evolutionary origin of structural colors

The first image-forming eyes in the fossil record of animals were discovered in the early Cambrian, about 530 million years ago. Large compound eyes are found in Cambrian fossils of trilobites which are a now extinct group of Arthropoda (Land and Nilsson, 2002). The evolution of the first eyes suddenly enabled animals to perceive their environment in terms of sizes, shapes, colors and behavioral patterns regardless of their status as predator or prey. Since then light perception has been one of the dominant origins for evolutionary pressure on the animals that led to diversification and adaptation to different ecological niches. The Arthropoda, as only one example, evolved hard external parts as defenses, which were even stable enough to survive in the fossil record through the geological time scale. Based on these hypotheses, some scientists even consider the evolution of the first image-forming eye to be the reason for the Cambrian Explosion (Parker, 2000; Parker, 2003; Parker, 2005). In response to the significant selection pressures generated by the evolution of visual perception, animals began to evolve pigments and photonic structures to produce colors used for signaling, camouflage or warning soon after the first eyes (Parker, 2005). In contrast to pigmentary colors, iridescence is a feature unique to structural colors. The additional information that can be delivered by changes in hue and/or intensity of the reflected light with different viewing angles is rich, which makes structural colors particularly suitable for intra- and interspecies visual communication (Parker, 2000; Doucet and Meadows, 2009).

2.2. Structure and chemical composition of insect cuticle

Insects represent not only the largest group within the Arthropoda, but they also evolved the highest number of species in the whole animal kingdom. Like all other arthropod species, the body of insects is - 7 -

Structure and chemical composition of insect cuticle completely covered by the cuticle which forms the exoskeleton of the animal. The main function of cuticle is to provide the shape and stability of the animals’ body, enable movement through the formation of joints and attachment sites for muscles, and to serve as a permeability barrier to the environment. In order to fulfill these functions, the cuticle forms skeletal elements that can have different physical properties which are brought about by local modifications of their structure and composition. The functions of skeletal elements are very diverse, like organs for sensing, respiration and color production (Neville, 1975) just to name a few. Insects pass through a series of growth stages (instars) separated by a series of molts until they become adult. The body structures of juvenile and adult instars of some groups of insects are slight different (ametabolous development) or broadly resemble each other except for the formation of wings and complete genitalia in adult instars (hemimetabolous development). Beetles largely change their form and habits from juvenile to adult instars (holometabolous development) and the final juvenile instar known as pupa is specialized to facilitate these changes (Gillott, 2005). The color generating cuticular structures of adult beetles are formed during the molting cycle between pupa and adult stage. The cuticle formation starts with the secretion of a new epicuticle by the epidermal cells, dissolution and re-absorption of the old endocuticle and the formation of a new exocuticle. This process is then followed by shedding off the old cuticle (ecdysis) and forming a new endocuticle post-ecdysially. Concurrently related processes include pre-ecdysial wax secretion and post-ecdysial tanning of the new cuticle (Neville, 1975; Gillott, 2005). The completely formed cuticle then consists of epicuticle, exocuticle and endocuticle from the distal to proximal in the sequence of their formation (Fig. 2.2.1; VII). The essential aspects of the structure and chemical composition of insect cuticle are described in the following paragraphs. Epicuticle The epicuticle is the outermost layer of insect cuticle (Fig. 2.2.1 VII, epi). It generally consists of two layers: an outer epicuticle and an inner epicuticle (Weis-Fogh, 1970; Wigglesworth, 1975). The epicuticle is mainly formed by a tightly cross-linked network of lipids

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Chapter 2 | Biological and Physical Background and proteins, which resists most methods of degradation (Andersen, 1979). In most insect species, the epicuticle is covered by two extracuticular layers, a lipid layer (wax layer) and, external to this, a cement layer (Lockey, 1988). The cement layer is produced by the dermal glands (modified epidermal cells) after ecdysis to protect the underlying wax. The outer epicuticle is the first layer of the new cuticle to be secreted. The chemical composition of the outer epicuticle is believed to be a mixture of lipids and a small amount of proteins which are hardened by oxidation and polymerization (Lockey, 1988). The outer epicuticle is resistant to the enzymes in the molting fluid even when it is newly secreted. Its main function is to protect the newly secreted exocuticle underneath from enzymatic digestion but meanwhile forms pores with diameters of about 3 nm letting through the digested products of the old endocuticle for re-absorption (Neville, 1975). The inner epicuticle forms the bulk of the epicuticle with the thickness ranging from 0.5 to 2 µm. It is composed of structural lipid (a lipoprotein) which is subsequently sclerotized (Neville, 1975; Lockey, 1988). Sclerotization or tanning is a complex process that stabilizes the proteinaceous structure through cross-linking of quinones with the functional groups of the proteins. The proteins covalently bonded by quinone tanning (sclerotins) are generally very tough and resistant to chemical and physical degradation (Hopkins and Kramer, 1992). The inner epicuticle is optically isotropic, while the outer epicuticle is birefringent. The refractive index of the inner epicuticle (1.58) is higher than those of dry proteins (1.54) or lipids (1.49), probably due to the presence of polyphenols (Weis-Fogh, 1970). Multilayer reflectors consisting of alternating electron-dense and -lucent layers have been observed in the transmission electron microscopy (TEM) micrographs of the inner epicuticle of tiger beetle species, where the electron-dense layers were suggested to be formed by melanoprotein granules with a refractive index of 2.0 (Schultz and Rankin, 1985b; Schultz and Rankin, 1985a). Similar multilayer structures were also found in the epicuticle of a leaf beetle (Plateumaris sericea) (Kurachi et al., 2002) and a jewel beetle (Chrysochroa fulgidissima) (Hariyama et al., 2005; Kinoshita, 2008), which are responsible for their coloration.

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Structure and chemical composition of insect cuticle

The inner epicuticle is pervaded by epicuticular channels (wax canals) with about 6 nm diameter which are fused into pore canals in the underlying exo- and endocuticles (Locke, 1961). The extractable lipids in the wax layer are secreted via these wax canals from the epidermis just before ecdysis (Neville, 1975; Andersen, 1979). The refractive index of the wax layer covering the dragonfly wings was determined to be 1.40 (Hooper et al., 2006).

Figure 2.2.1: Structural hierarchy of the chitinous organic matrix in arthropod cuticle. N-Acetylglucosamine molecules (I) form anti-parallel chains of α-chitin (II). Protein-coated chitin crystallites form nanofibrils (III) that assemble to chitin/protein fibres (IV) arranged in horizontal planes (V). The planes stack helicoidally forming the twisted plywood structure (VI) constituting the bulk cuticle (VII). (VII, epi) depicts the typical organization of the epicuticle including the pore canal system. Procuticle The procuticle is secreted by epidermal cells after the epicuticle. It forms the mechanical relevant part of the cuticle and can be further divided into two layers: exocuticle and endocuticle (Hadley, 1986). In contrast to the chitin free epicuticle, the procuticle is composed of

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Chapter 2 | Biological and Physical Background chitin crystallites embedded in a protein matrix. The exocuticle is deposited before ecdysis and the proteins inside are sclerotized afterwards, which usually leads to a brown or black color appearance. The endocuticle is not sclerotized, and thus can be digested by enzymes present in the molting fluids (Neville, 1975) to recycle at least a part of the components of the old cuticle. From a material science point of view, the chitinous procuticle is a hierarchically structured fibrous composite material (Fig. 2.2.1). At molecular level, chitin consists of polysaccharide chains (Fig. 2.2.1 II) of N-acetyl-ß-D-glucosamine (Fig. 2.2.1 I). Several such chains (about 18 to 25) are associated together in an ordered manner via hydrogen and covalent bonding to form a chitin crystallite of 2.8 nm in diameter and about 300 nm in length (Neville et al., 1976). Chitin crystallites usually exist in three polymorphs, α-, β-, and γ-chitin, inside which the molecular chains are arranged in a parallel, antiparallel or combined fashion, respectively (Rudall, 1963). α-chitin is the major component of arthropod cuticle and also the most abundant form of chitin crystallite in nature. The unit cell of α-chitin is orthorhombic with the measured lattice constants a, b and c amounting to 4.74 Å, 18.86 Å and 10.32 Å, respectively, where the c axis corresponds to the longitudinal direction of the chitin crystallite (Minke and Blackwell, 1978). The chitin crystallites are wrapped by proteins arranged probably in a six-fold helicoidal fashion forming chitin/protein nanofibrils with a diameter of 7.25 nm (Blackwell and Weih, 1980) (Fig. 2.2.1 III). In some cases, a number of such nanofibrils cluster to form thicker chitin/protein fibers (Fig. 2.2.1 IV) which are aligned parallel to each other forming the horizontal chitin/protein fiber planes (Fig. 2.2.1 V) parallel to the cuticle surface. The fiber directions gradually rotate in the successively deposited planes around the normal direction of the plane and repeat after each rotation of 180°. This configuration of chitin/protein planes forms an anticlockwise (left handed) helicoidal structure in almost all studied cases which is also termed as twisted plywood structure (Fig. 2.2.1 VI) or Bouligand structure (Bouligand, 1965; Neville, 1975; Giraud-Guille, 1998). This structure is analogous to the helicoidal microstructure in cholesteric liquid crystals (Neville and Caveney, 1969). Due to the nature of the plywood structure and the inherent curvatures of the bulk cuticle, a number of different patterns can be observed in samples prepared for

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Structure and chemical composition of insect cuticle microscopical investigation, like lamellae formed by a series of arc shaped patterns at oblique section planes (Neville, 1975; Giraud- Guille, 1998) (see the patterns in the exo- and endocuticle regions in Fig. 2.2.1 VII). These lamellae sometimes appear to be arranged obliquely with respect to the cuticle surface (Weis-Fogh, 1970), which was explained by Bouligand (1972) with the fibers building the helicoid being not straight but curved (Fig. 2.2.2a).

Figure 2.2.2: (a) Schematic drawing of helicoidal structure formed by rotation of curved rather than straight chitin/protein nanofibrils. The apparent lamellae of arc patterns traverse obliquely to the planes (reprinted from Bouligand, 1972, with permission from Elsevier). (b) Model showing that the arc patterns originate from oblique sections of pore canals resembling twisted ribbons (reprinted from Neville and Berg, 1971, courtesy of "The Palaeontological Association"). (c) Schematic drawing showing that the elliptical cross sections of the pore canals in (b) form the arc patterns coinciding with the arc patterns resulting from the rotation of fibers (reprinted from Neville and Berg, 1971, courtesy of "The Palaeontological Association"). Another important structural system inside procuticle are the pore canals which pervade vertically through the procuticle as extensions of the epidermal cells and function as a transport system for materials (Wigglesworth, 1948; Neville, 1975). Due to the rotation of the chitin nanofibrils or, if formed, chitin/protein fibers which are perpendicular to the pore canals, the shape of these canals resembles twisted ribbons (Fig. 2.2.2b). The long axis of the elliptical cross section of each canal is orientated in the same direction as the surrounding fibers. Thus, upon obliquely cutting through cuticle, the parabolic patterns of the

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Chapter 2 | Biological and Physical Background profiles of pore canals coincide with the arcs shaped patterns resulting from the rotation of fibers (Fig. 2.2.2c) (Neville, 1975). The helicoidal structure in the exocuticle of many Scarab beetle species were regarded as an optical analogue to cholesteric liquid crystals that can selectively reflect left handed circularly polarized light within a certain wavelength range (Neville and Caveney, 1969; Goldstein, 2006; Jewell et al., 2007; Sharma et al., 2009). The cuticular materials of these beetles are often optically anisotropic, e.g. for Potosia speciosissima the refractive indices parallel and perpendicular to the cuticle layer are = 1.580 and = 1.598, respectively (Caveney, 1971). All species of Plusiotis examined by ∥ ⊥ Caveney and several species of Anoplognathus푛 have uric acid푛 in their exocuticle, which increases the birefringence of the system (e.g. for Plusiotis optima = 1.600 and = 1.684) thus enhances the reflectivity of the cuticle (Caveney, 1971). There are also Scarab ∥ ⊥ beetle species like푛 Plusiotis resplendens푛 that reflects both left and right circularly polarized light at different wavelength ranges due to the change of polarization state by the unidirectional orientated layer acting as a half-wave plate sandwiched between two helicoidal layers (Caveney, 1971). In contrast, Heterorrhina species do not selectively reflect circularly polarized light of any handedness. Their exocuticle generally contains a well-developed vertical rod system (Neville and Caveney, 1969). The endocuticle of some insect species in the pupal or adult stage acts as a broad bandwidth multilayer reflector generating a metallic gold or silver color (Neville, 1977; Parker et al., 1998; Vigneron et al., 2007). Parker et al. (1998) suggested that the progressive change in the lamellar spacing of the endocuticle forms a multilayer reflector which reflects most components of the incident white light resulting in a gold or silver color. Two tortoise beetle species (Aspidomorpha tecta and Charidotella egregia) were reported to change their colors when disturbed by external stress, which is realized by changing the pH and water content in the endocuticle by the epidermis (Neville, 1977; Vigneron et al., 2007).

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Structure and chemical composition of insect cuticle

Cuticular scales In general, scales are outgrowths of the cuticle that cover the body and wings of insects. Scales are reported to appear in silverfish species, an ancient group of Arthropoda. These scales can reflect infrared light (Large et al., 2001). In the scales of butterfly and moth species, various forms of photonic structures exist which generate vivid colors and different optical effects (Ghiradella, 1989; 1991; 1994; Vukusic et al., 1999; Vukusic et al., 2001; Lawrence et al., 2002; Vukusic et al., 2002; Stavenga et al., 2004; Vukusic et al., 2004; Kertesz et al., 2006; Prum et al., 2006). Color-producing photonic structures were also found in the scales of different beetle species, such as longhorn beetles (Liu et al., 2009; Dong et al., 2010; Dong et al., 2011; Simonis and Vigneron, 2011; Colomer et al., 2012) and weevils (Parker et al., 2003; Welch et al., 2007; Galusha et al., 2008; Pouya et al., 2011; Wilts et al., 2011; Wilts et al., 2012; Wu et al., 2013). Among various other structures, many butterfly and weevil species (Michielsen and Stavenga, 2008; Saranathan et al., 2010; Wilts et al., 2011; Wilts et al., 2012; Wu et al., 2013) have evolved photonic crystal structures geometrically corresponding to so-called bicontinuous cubic structures (BSCs). Three fundamental BSCs, P-, D- and G-surface structure (Fig. 2.2.3), can be described by dividing the volume of cubic structures into two complementary networks by triply periodic primitive, diamond and gyroid surfaces, respectively (Michielsen and Stavenga, 2008). The G-surface structure (also called gyroid structure) usually occurs in the scales of butterflies (Michielsen and Stavenga, 2008; Saranathan et al., 2010), which can selectively reflect circularly polarized light (Saba et al., 2011), while the D- surface structure occurs in the scales of weevil beetles (Wilts et al., 2011; Wilts et al., 2012; Wu et al., 2013). How these structures are formed by the insects is largely unknown. There is only one study available where Ghiradella (1989) shows that the gyroid structure in the scales of a butterfly species (Callophrys gryneus) is formed by the deposition of cuticular material extracellularly on a 3D template molded by the plasma membrane whose structure is in turn based on complex invaginations of the intracellular smooth endoplasmic reticulum. To present, there is no study available on the development of three-dimensional photonic crystals in the weevil scales.

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Chapter 2 | Biological and Physical Background

Figure 2.2.3: Models of the unit cells of three fundamental bicontinuous cubic structures: (a) D-surface structure, (b) G-surface structure, (c) P- surface structure.

2.3. Physical background of photonic structures occurring in nature

2.3.1. Photonic crystals

Color-generating biological photonic structures are usually composed of dielectric materials of which the refractive index (n) varies periodically on a length scale comparable to the wavelength of light. Light propagating in these photonic crystals is diffracted by different lattice planes and the interference of these diffracted waves determines the optical properties of the structure. This mechanism was well- studied for X-ray diffraction of atomic crystals, where X-ray reflection occurs due to constructive interference if the Bragg condition is met (James, 1948). The Bragg diffraction therefore is associated with a photonic stop gap meaning that electromagnetic waves with frequencies in this gap cannot propagate through the crystal. The amplitude of the waves of these frequencies decays exponentially into the crystals. The size of the stop gap (the so called photonic interaction strength ψ) is defined as ∆ω/ωm, where ∆ω is the gap’s frequency width and ωm is the middle frequency of the gap. The Bragg length (LB=2d/(πψ)) is the characteristic length of the exponential decay (Vos et al., 1996a; Vos et al., 1996b; Koenderink, 2003), where d is the interplanar distance. The gap size is proportional to the refractive index contrast (∆n/ ) of the photonic crystal, where ∆n is the difference between the refractive indices of constituting phases 푛� - 15 -

Physical background of photonic structures occurring in nature and is the volume-average refractive index of the entire structure. The spatial distribution of the phases in the photonic crystal also has an in푛�fluence on the gap size (also see Section 2.3.4) (Yablonovitch, 1993; Lopez, 2003; Joannopoulos et al., 2008). Since the refractive index contrast for X-ray is very small (~ 10-4), the allowed frequency range of X-ray to be Bragg diffracted by atomic crystals is very narrow (Yablonovitch, 1993). The index contrast for photonic crystals is much larger (~ 0.5), hence the gap size is larger and the light with the frequency in the stop gap can penetrate only a few lattice planes into the crystal. Essentially, it is the nonzero index contrast of photonic crystals that relaxes the Bragg condition allowing a wider frequency range of light to be reflected (Koenderink, 2003). In three dimensions, the so-called complete photonic band gap is achieved if the gap sizes in all directions are large enough to overlap with each other. Possession of photonic band gaps is the most important feature of photonic crystals. Photonic band gaps are an analogue to the energy band gaps of semiconductors in solid-state physics. Photonic crystals can thus form perfect optical “insulators”, which can guide light without losses in lower-index media and confine light within nano- cavities, among other novel possibilities for control of electromagnetic phenomena (John, 1987; Yablonovitch, 1987; Soukoulis, 2001; Akahane et al., 2003; Russell, 2003; Eleftheriades and Balmain, 2005; Joannopoulos et al., 2008).

2.3.2. Multilayer structures One-dimensional photonic crystals consisting of alternating layers with different refractive indices (Fig. 2.3.1) are the simplest type of photonic crystals. A multilayer structure can act as a Bragg mirror which selectively reflects light with different frequency ranges (Joannopoulos et al., 2008). In nature, most of the known structural colorations originate from this type of structure or its analogues (Kinoshita, 2008). The first to study electromagnetic wave propagation in the one-dimensional photonic crystal was Lord Rayleigh (1887; 1917). Wavelength-dependent reflectivity of multilayer structures is now often calculated either using the transfer matrix method by solving Maxwell’s equations (Hecht, 2002) or by Huxley’s method (Huxley, 1968).

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Chapter 2 | Biological and Physical Background

Figure 2.3.1: Model of a one-dimensional photonic crystal consisting of two alternating layers A and B with different refractive indices nA and nB and different thicknesses dA and dB. θ is the angle of incidence of light and d = dA + dB is the structural periodicity of the system. The reflection of multilayer structures can also be understood as a result of the presence of a photonic band gap generated by a one- dimensional photonic crystal. The respective calculations based on the plane-wave method will be introduced in Section 2.3.4. Here, a simple method for the estimation of the wavelength of reflected light is given. This method assumes the multilayer structure as a semi-infinite one- dimensional periodic stack with weak refractive index contrast. Under this assumption, the dominant reflected wavelengths can be determined based on a long wavelength approximation (Vigneron et al., 2006). Long wavelength approximation means the light with long wavelengths doesn’t probe the fine structure of the crystal. Instead, the light effectively sees a homogeneous dielectric medium, with an effective refractive index that is a weighted average of the refractive indices nA and nB of both constituting phases (Joannopoulos et al., 2008). The value of is푛� confined in the Weiner bounds (Aspnes, 1982). For a composite consisting of two materials, it is bounded by 푛� (2.3.1) + −1⁄2 + 1⁄2 퐴 퐵 퐴 퐵 where d , 푑d are−2 the푑 thicknesses−2 of individual푑 layers2 푑 and2 d = d + d is A� B 푛퐴 푛퐵 � ≤ 푛� ≤ � 푛퐴 푛퐵� A B the structural푑 periodicity푑 of the system. The푑 photonic푑 band gap is then formed at wavelength λ calculated by Eq. 2.3.2

- 17 -

Physical background of photonic structures occurring in nature

2 sin = (2.3.2) 2 2 which is similar to Bragg’s law,푑√ 푛�where− θ is휃 the incidence angle in air 휆 and m takes integer numbers. For normal푚 incidence Eq. 2.3.2 is simply 2 = (2.3.3) 푑푛� 휆 2.3.3. Helicoidal photonic structures푚 The optical properties of helicoidal photonic structures as found in the exocuticle of Scarab beetles are anisotropic, which is different from the isotropic properties of the multilayer structures located normally in the epicuticle of other beetle species. The experimental results (Section 2.2) have shown that the refractive index parallel to the cuticle surface ( ) of Scarab beetles is smaller than the one perpendicular to it ( ) (Caveney, 1971). The exact optical properties ∥ of single chitin/protein푛 fibers that form this bulk birefringent material ⊥ are unknown. If one푛 assumes that the individual chitin/protein fiber is a uniaxial birefringent material of which the extra-ordinary refractive index ( ) along the longitudinal direction of the fiber is different from the ordinary refractive index ( ) perpendicular to the fiber (Fig. 푒 2.3.2), light푛 propagating along the helicoidal axis (z axis in Fig. 2.3.2) 표 would experience refractive indices푛 varying continuously from to with increasing depth due to the successive rotation of fiber planes 표 perpendicular to it (Fig. 2.3.2). The periodicity of this variation푛 of 푒 푛refractive index is one half of the pitch (p), which corresponds to 180° rotation of the fibers.

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Chapter 2 | Biological and Physical Background

Figure 2.3.2: Model of a left handed helicoidal photonic structure with a pitch of p, which can selectively reflect left circularly polarized light. The fibers forming this structure consist of a uniaxial birefringent material of which the extraordinary refractive index ne is different from the ordinary refractive index no. θ is the angle of incidence of light. This biological helicoidal structure resembles the in plane molecular rotation of cholesteric liquid crystals. The wavelength of maximum reflection (λ) of this periodic structure thus can also be calculated by the equation sin = (2.3.4) 2 2 similar to Bragg’s law applied푝√푛� −for liquid휃 crystals, where = 휆 ( + 2 ) 3 is the geometric 푚average refractive index of the 푛� helicoidal2 structure,2 θ is the incidence angle in air and m takes integer 푒 표 �numbers푛 (Fergason푛 ⁄ , 1966; Dreher and Meier, 1973; de Gennes and Prost, 1995). Similar to cholesteric liquid crystal, the anticlockwise rotating helicoidal structure in the exocuticle of Scarab beetles is able to selectively reflect circularly polarized light, in most cases left

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Physical background of photonic structures occurring in nature circularly polarized light (Neville and Caveney, 1969; Caveney, 1971).

2.3.4. 3D photonic crystals The most widely used theoretical approach to study the light propagation in three-dimensional (3D) photonic crystals is the plane- wave expansion method. The photonic crystals for investigation normally are assumed to consist of lossless (non-absorptive) and linear dielectric materials. Maxwell’s equations for an inhomogeneous dielectric medium without charges (ρ = 0) and currents (J = 0) are

× = (2.3.5) 휕푩 ∇ ×푬 =− (2.3.6) 휕푡 = 휕0푫 (2.3.7) ∇ 푯 = 0휕푡 (2.3.8) where E and H are the electric∇ ∙ and푩 magnetic fields, D and B are the electric and magnetic inductions,∇ ∙ 푫 respectively. Combine Eqs. 2.3.5-8 with the constitutive relations for non-magnetic materials = ( ) (2.3.9) = ( ) (2.3.10) 0 where ε 0 and µ 0 are the vacuum푫 휀 휀permittivity풓 푬 and permeability, ε (r) 0 are the dielectric function (also푩 휇called휇 풓 relative푯 permittivity), and µ (r) are the relative permeability (µ (r) ≈ 1), one can deduce the wave equations for the magnetic and electric field as 1 × × ( ) = ( ) (2.3.11) ( ) 2 휔 1 ∇ � ×∙ ∇ ×푯(풓)� = � � 푯( 풓) (2.3.12) ( ) 휀 풓 푐 2 휔 where ω is the angular∙ ∇ �frequency∇ 푬 풓 �and� c �is 푬the풓 speed of light in vacuum. These 휀are풓 two eigenvalue problems푐 for Maxwell’s equations and Eq. 2.3.11 is preferred for numerical calculations for the reason of mathematical convenience (Joannopoulos et al., 2008). Because of the periodicity of the dielectric functions in photonic crystals, the eigenmodes ( ) in the eigenvalue problems (Eq. 2.3.11) can be expanded in plane waves 푯풌 풓 - 20 -

Chapter 2 | Biological and Physical Background

( ) = ( ) (2.2.13) according to Bloch’s theorem, where푖풌∙풓 ( ) is a function with the 풌 풌 periodicity of the crystal푯 lattice풓 푒 and풖 k 풓is a Bloch wave vector in 풌 reciprocal space (Kittel, 2005; Joannopoulos풖 풓 et al., 2008). In order to solve the eigenvalue problems, one expands the Bloch modes and inverse dielectric constant (η = 1/ε) in a Fourier series in terms of the reciprocal lattice vector G ( ) ( ) = (2.3.14) 풌 푖 풌+푮 ∙풓 푯풌 풓 1� 풖푮푒 ( ) = 푮 = ( ) (2.3.15) 푖푮∙풓 Insert these two Fourier휂 풓series (Eq. 2.3.14� 휂푮 and푒 2.3.15) into the wave 휀 풓 푮 equation for the magnetic field (Eq. 2.3.11), an infinite set of linear eigenvalue equations is deduced as ( ) ( + ) × [( + ) × ] = 2 (2.3.16) ′ ′ 풌 휔 풌 풌′ This �linear휂푮 −푮eigenvalue풌 푮 problem풌 푮 is 풖the푮 basis� for� 풖the푮 numerical 푮 푐 approximations to the electromagnetic eigenmodes and the dispersion relations of photonic crystals, known as plane-wave expansion method (Ho et al., 1990; Sözüer et al., 1992; Joannopoulos et al., 2008). The dispersion relation of a homogeneous material is ω = ck/n (the straight dashed line in Fig. 2.3.3a) where k is the wavevector and n is the refractive index of this material. For the simple one-dimensional photonic crystal with the same average refractive index n and period d as shown in Fig. 2.3.1, frequency stop gaps (yellow stripes in Fig. 2.3.3a) open up when the Bragg condition k = ± G/2 = ±mπ/d is met, where G = 2πm/d is a reciprocal lattice vector and m takes an integer. The first stop gap occurs at k = -π/d and k = π/d, between which is the first Brillouin zone defining a region in reciprocal space closer to the origin than to any other reciprocal lattice points. Other stop gaps occur for lager integer m at higher frequency regime, but can be plotted also in the first Brillouin zone by subtracting some reciprocal lattice vector mG from the dispersion curves due to the periodicity of the photonic crystal (Fig. 2.3.3a).

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Physical background of photonic structures occurring in nature

Figure 2.3.3: (a) Schematic drawing for the dispersion relations for a homogenous material (dashed line) and for the one-dimensional photonic crystal as shown in Fig. 2.3.1 (modified based on reprint from Lopez, 2003, with permission from WILEY-VCH). ω is the light frequency, k is the wavevector, d is the structural periodicity of the one-dimensional photonic crystal, and G is the reciprocal lattice vector. (b) The first Brillouin zone of the face-centered cubic (f.c.c.) structure and the traditionally labeled high symmetry points. The yellow polyhedron indicates the smallest region in the Brillouin zone for which the dispersion relations are not symmetry related (irreducible Brillouin zone). This construction of the dispersion relation of photonic crystals results in the often plotted photonic band structure diagrams used for predicting the frequencies and sizes of the band gaps determining the optical properties (e.g. Fig. 2.3.4). Since for photons there is no length scale involved as the Bohr radius for atomic physics, the lattice constant of the photonic crystal a is taken as the fundamental length scale, thus the frequency is usually plotted in the units of c/a. This lack of an absolute length scale means that the properties of the photonic crystals are lengthwise scalable, i.e. resize the crystal (changing of a) will only change the unit of the frequency (c/a) but not the value of the normalized frequency itself. Therefore, the photonic band structure calculated for photonic crystals operating in the visible wavelength range can be used for the photonic crystals with the same structure and refractive indices but larger dimension in microwave regime or smaller one in ultraviolet regime as well. The x- axis of the diagrams represents the linear trajectory through the origin Γ and high symmetry points located at the surface of the first Brillion zone in the reciprocal space. The first Brillouin zone of a f.c.c.

- 22 -

Chapter 2 | Biological and Physical Background structure is a truncated octahedron as shown in Fig. 2.3.3b. The traditionally labeled symmetry points Γ, L, X, K, U and W in Fig. 2.3.3b are also labeled in the band structure diagrams of different diamond-based structures in Fig. 2.3.4. In these diagrams, the panels ΓL and ΓX show dispersion relations with increasing wavevector k in <111> and <100> directions, respectively. In panels such as XW, the modulus of k increases and the direction of k changes from <100> to <210> continuously (Lopez, 2003; Joannopoulos et al., 2008). The band structure diagram of a diamond lattice of dielectric spheres in air (Fig. 2.3.4a) shows narrow stop gaps along different directions between bands 2 and 3, when the refractive index of the spheres is relatively low (nsphere = 1.56). If the refractive index of the spheres is higher (nsphere = 12), i.e. ∆n between the spheres and air is larger, the diagram of a similar diamond based structure (Fig. 2.3.4b) with the same volume fraction√ of spheres (Vf = 50 %) shows wider stop gaps overlapping with each other forming a complete band gap with the size (∆ω/ωm) of 10.9 %. Lower volume fraction of the spheres (Vf = 35 %), consequently decrease , broadens the band gap to 11.3 % (Fig. 2.3.4c). Comparisons of the band structure diagrams of the above three similar structures show that푛� the size of the band gaps largely depends on the ratio of ∆n/ as mentioned in Section 2.3.1. However indefinitely increasing ∆n by increasing nsphere will not broaden the gap to an arbitrary size, since 푛increases� simultaneously. Decreasing by designing a system with a very low volume fraction of the spheres will not lead to a wider gap as푛 �well, since the gap size is also proportional푛� to the fraction of the electric-field energy of the light in these regions (Joannopoulos et al., 2008). When the symmetry and constituting materials of two photonic crystals are the same, the spatial distribution of these materials has a significant influence on the gap size. For example, the so-call rod connected diamond structure (Fig. 2.3.4d) possesses as almost twice as large band gap compared to the diamond lattice of spheres (Fig. 2.3.4c) constructed using the same amount (Vf = 35 %) of the materials with equal refractive index (nrod = nsphere = 12). Optimizing the photonic band gap size by varying the geometry and the filling fractions in the unit cell of the photonic crystals, while √keeping the crystal symmetry and the refractive indices of the constituting materials unchanged, is one of the intensively studied topics in the field of photonic crystals (Maldovan and Thomas, 2004).

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Physical background of photonic structures occurring in nature

Figure 2.3.4: Photonic band structure diagrams of four types of photonic crystals with diamond-based structure: (a-c) Diamond lattice of spheres in air with different volume fractions of the spheres (Vf) and different refractive indices for the spheres (nsphere); (d) Rod connected diamond structure with the same Vf and refractive index for the constituting materials nrod = nsphere as in (c).

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Chapter 3 | Materials and Methods

Chapter 3. Materials and Methods

3.1. Materials

The beetles used for this study are dry museum specimens obtained from the entomological collections of the Staatliches Museum für Naturkunde Stuttgart courtesy of Dr. Joachim Holstein, who also helped in determining the species. These beetles are two Ground beetle species (Carabus auronitens and Carabus auratus), two Scarab beetle species (Cetonia aurata and Dicranorhina oberthuri) and one weevil species (Entimus imperialis).

3.2. Sample preparation

The millimeter sized samples of the four Ground and Scarab beetle species used for optical characterization, chemical treatment and for further ultra-microtome preparation were dissected from their elytra. Samples used to characterize the structure of the broken cross- sectional surfaces of the elytra were laterally fractured using tweezers. The micrometer sized scales of the weevil species were collected by scraping them from the surface of the cuticle with a needle. Scales for exposing the structured core by focused ion beam (FIB) milling were spread and glued to the surface of self-adhesive carbon pads. The scales for microtome polishing were spread on the surface of a drop of super glue and thus fixed. After curing, the top surfaces of the partially embedded scales were sectioned. The scales for characterizations of their cross section by microtome polishing were not scraped from the elytra, but were fixed on the elytra by emerging them in-situ using super glue. To ensure a mechanically stable fixation of the scales, the elytra were put into vacuum for 20 minutes to eliminate the air gaps between the scales and glue. All samples for ultra-microtome preparation were first glued to cylindrical plastic holders. Then they were polished with an ultra-

- 25 -

Structural characterization microtome (PowerTome PT-XL, Reichert-LABTEC) equipped with a diamond knife by sequentially decreasing the section thickness from 500 nm to 20 nm. Aqueous NaOH solutions were used to selectively remove cuticle components like proteins and lipids. All NaOH treatments were performed at room temperature and under continuous stirring using a magnetic stirrer. After the chemical treatment, the samples were washed in distilled water for a few seconds followed by dehydration in pure methanol for a few seconds.

3.3. Structural characterization

The cuticular microstructure was characterized in detail by scanning electron microscopy (SEM, Zeiss Gemini 1540 XB, Oberkochen, Germany) using low acceleration voltage (5 keV), a small aperture (30 µm) and small working distances (2~4 mm) for maximum resolution and low beam damage. The samples for SEM analysis were rotary shadowed with 2 nm platinum using a precision etching coating system (Model 682, Gatan Inc.). During the process, the thickness of coating was controlled using a thickness monitor (Model 681, Gatan Inc.). In order to expose the photonic crystals in the core of scales of weevil E. imperialis for SEM observation, the shell covering these photonic crystals was removed by FIB milling using built-in FIB column (Orsay Physics, Fuveau, France) in SEM. The current of the Ga ion beam for milling is 500 pA and the accelerating voltage was 30 kV. The scales were first milled perpendicularly with respect to the top surface (vertical gray arrow in Fig. 3.3.1) to expose their cross sections. Guided by the interface between the shell and core revealed by the already exposed cross sections, the scales were subsequently milled parallel to the top surface (horizontal gray arrow in Fig. 3.3.1) until the shell was removed.

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Chapter 3 | Materials and Methods

Figure 3.3.1: Schematic drawing of the FIB milling procedure to expose the photonic crystals in the scales (see also Fig. 6.2.6).

3.4. Chemical characterization

In order to determine the chemical composition of the second solid phase discovered in the transparent domains of scales of E. imperialis, energy dispersive X-ray (EDX) spectra and infrared spectra of both transparent and colored domains were recorded. The EDX spectra were recorded using an EDAX system (AMETEK GmbH, Wiesbaden, Germany) built in the same SEM for structural characterization (see Section 3.3). The applied accelerating voltage varied from 5 to 12 kV, the aperture is set to 60 µm and the working distance is about 10 mm. For measurement of Fourier transform infrared spectra (FTIR, Bruker Hyperion 3000 IR microscope on Vertex 70v spectrometer), individual scales were placed on a double sided polished piece of germanium measuring 2 × 4 cm. Spectral images were measured in transmission geometry on the microscope's 64 × 64 pixel focal plane array detector, and referenced against a region of the germanium piece without scales.

3.5. Optical characterization

The color and surface microstructure of samples were characterized by using optical microscope (OM, Leica DM 4000M) in bright field mode. The white balance for the microscope was adjusted against a white paper before taking the color pictures. The smallest aperture of

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Optical characterization the microscope and highest intensity of the incident light was used in order to represent the original color and clear microstructure of the samples in the best possible way. Polarizer and analyzer can be inserted into the OM to adjust polarization state of the incident and reflected light, respectively. Cross or parallel polarization is achieved by setting the polarization state of polarizer and analyzer perpendicular or parallel to each other, respectively. Reflectivity spectra of samples at normal incidence were obtained using an OM (Aristomet, Leica) equipped with a visible spectrometer (CCS200, Thorlabs). The spectral range of the spectrometer is 210 – 1100 nm. The measurable spectral range covers the entire visible spectral range, but is not as wide as the theoretical range of the spectrometer due to the constraint of the optics of the microscope. Raw light intensities were measured under objectives of different magnifications with different numerical apertures (NA): 5 × (NA = 0.15), 10 × (NA = 0.25), 20 × (NA = 0.45), 50 × (NA = 0.85) and 100 × (NA = 0.90). To measure the spectra of single colored domains in scales of E. imperialis, the areas of detection were narrowed down to about 30 µm in diameter by an adjustable iris in the OM. Reflectivity spectra of the elytra of both Ground beetle and Scarab beetle species at different angle of incidence were obtained on a Sentech SE800 spectroscopic ellipsometer (Sentech GmbH, Berlin/Krailling, Germany), with the compensator deactivated, and both polarizer and analyzer being set to either horizontal (p) or vertical (s) polarization. The available spectral range for the experiments was 300-820 nm. The incident light was focused to a spot diameter of ~200 µm with a microspot lens to the colored region of Ground beetle species avoiding the ridge region. The dark current of the CCD line detector was subtracted from all intensities before analysis. The spread in both illumination as well as detection angle due to the microspot lenses was ~4° each. The raw light intensities of samples measured by the last two experiments were converted into reflectivity (see Eq. 3.5.1) through the following procedure. First, an ellipsometric measurement was performed on a silicon wafer, followed by a measurement of its reflected raw intensities (ISi). The ellipsometric measurement was

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Chapter 3 | Materials and Methods modeled, and the resulting model was used to compute the reflectivity of the silicon wafer (RSi). From this reflectivity and the raw intensity reflectivity spectra, the product (A) of the response function of the optical system, the efficiency of the detector and the emission properties of the light source was calculated. This product was subsequently used to convert the measured raw intensities of samples (IS) into their reflectivities (RS). S Si S = , ( = ) (3.5.1) Si 퐼 퐼 Optical scattering 푅experiments of 퐴the elytra of Ground beetle species were conducted on a퐴 modified single푅 wavelength ellipsometer Multiskop (Optrel GbR, Sinzing, Germany) using a wavelength of 532 nm. The quarter-wave plate was removed from the system, and the detector was replaced by an optical power meter Nova II (Ophir Optronics, Jerusalem, Israel). Angular-dependent reflectivity curves were obtained by a concurrent movement of the incidence and detector arm. The sample position was adjusted so that the beam was always pointing to the same spot with an accuracy of ~1 mm. In the scattering experiments, the incidence arm was kept at a constant angle of incidence, while the detector arm was moving between a detection angle of 25° and 80°. All measured intensities were normalized by the incident intensities, which were obtained by placing the power meter before the sample. The angular spread of the detection angle was ~1°, as verified by measurement of the scattering curves from a flat silicon wafer.

3.6. Optical simulation

Reflectivity spectra of the elytra of Ground beetle species were calculated by using the open source software “reflcalc” (Erbe, 2008) based on the model abstracted from the microstructure of their epicuticle shown in SEM images. “Reflcalc” computes reflectance based on the first-order Maxwell equations using matrix methods (Lekner, 1987; Schubert, 1996). The photonic band structure of the 3D photonic crystals in the scales of E. imperialis was calculated by solving Maxwell’s equations for eigenmodes in the frequency-domain (see Section 2.3.4) using the

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Optical simulation

MIT Photonic-Bands (MPB) package (Johnson and Joannopoulos, 2001). An example of the script file that specifies the input dielectric function and output photonic band structure is given in Appendix. The refractive index of the cuticular network is assumed to be 1.56 (Vukusic et al., 1999), and the one for air is 1.00. The lattice constants of the photonic crystal in different domains were determined by measuring the dimensions of the solid photonic crystal network on the recorded SEM images. This biological photonic crystal was modeled by a D-surface structure model which can be approximated by a constant mean curvature surface modeled by a level surface: ( , , ) = cos sin( + ) + sin cos( ) = (3.6.1) where X = 2πx, Y = 2πy and Z = 2πz. (x, y, z) are the positional coordinates푓 푥 in푦 푧the crystal푍 structure.푋 푌 The푍 other푋 −two푌 fundamental푡 bicontinuous cubic structures, G- and P-surface structure (see sub- section Cuticular scales in Section 2.2), can also be modeled by level surfaces: ( , , ) = sin cos + sin cos + cos sin = (3.6.2) ( , , ) = cos + cos + cos = (3.6.3) The isovalue푓 푥 푦t determines푧 푋 the푌 volume푌 fraction푍 of푋 the 푍two 푡continuous networks divided푓 푥by푦 the푧 triply 푋periodic푌 D-, G-푍, P-surface푡 (Schnering and Nesper, 1991; Wohlgemuth et al., 2001; Michielsen and Stavenga, 2008). These models were visualized (see Fig. 2.2.3) using the open source software K3DSurf (Taha, 2012). The value of t in Eq. 3.6.1 for the photonic crystal of E. imperialis was adjusted by determining the ratio between the diameter of the holes and the thickness of the neck of the rods from SEM images (see Fig. 6.2.7b). The modeling of the structure and the calculation of the photonic band structure were tested and confirmed by matching the resulting band structures of all three fundamental bicontinuous cubic structures to the results in literature, e.g. Maldovan et al. (2002) and Saranathan et al. (2010).

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Chapter 4 | Multilayer Structures (Ground Beetles)

Chapter 4. Multilayer Structures (Ground Beetles)

4.1. Introduction

The Ground beetles (Carabidae) are a large group of beetles with about 30000 species worldwide and over 700 species alone in Central Europe. Many Carabus species are of dark coloration, but some of them display vivid coloration with a metallic sheen to it. The colors range from dark blue over purplish and copper tones to shiny green and golden tones. I investigated the origins of the green coloration of the two middle European Ground beetle species Carabus auronitens and Carabus auratus (Fig. 4.1.1). The coloration of Carabus auronitens (Fig. 4.1.1a) is very variable and partially depends on the environmental conditions of its habitat. The elytra of the investigated beetle appear greenish with a metallic sheen. Each elytron is decorated with three strong longitudinal ridges with dark, nearly black color. Between these the surface appears finely granulated. The animals live in humid and tempered deciduous and coniferous forests and are found mostly under loose bark, in dead wood and mossy areas. The elytra of C. auratus (Fig. 4.1.1d) are metallic green-golden in color. The three longitudinal ridges located on each elytron are broader, brighter and much less distinctive than those of C. auronitens and the fine granulation is missing. This thermophilic species lives in fields, dry grassland, and forest borders (Harde and Severa, 1988). To my knowledge, there is no report so far on the coloration mechanism of the metallic colors displayed by the cuticle of these two beetle species. In my master studies, I investigated the structure and composition of the cuticles of these two species via selective removal of materials with chemical treatment to characterize their greenish elytra. These results will be briefly recalled here, since they are the base of further structural characterization, optical experiments and theoretical modeling, which explain the optical mechanisms responsible for the coloration of the cuticles of these two species.

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Structural characterization

Figure 4.1.1: Morphology of the two Carabus Ground beetle species investigated in this study: C. auronitens (a) and C. auratus (d). Optical microscopy (OM) images show that the color regions of the elytra from C. auronitens (b) and C. auratus (e) have different colors. The transparent surface layers (TSL in b, e) modify the color of the cuticle to shorter wavelengths. The ridge regions (c, f) of both beetles display a similar brownish color.

4.2. Structural characterization

4.2.1. Top surfaces of original samples In terms of optical appearance, the elytra of the two species can be divided into two regions: color regions (Fig. 4.1.1b, e) and dark ridge regions (Fig. 4.1.1c, f). The color regions are covered by an incomplete transparent surface layer (TSL in Fig. 4.1.1b, e) which influences the visible color. The uncovered areas of C. auronitens display yellow-green color (Fig. 4.1.1b) whereas the uncovered areas of C. auratus show golden color with some locations having a more reddish hue (Fig. 4.1.1e). In both species, the areas covered by the transparent surface layer reflected light of shorter wavelengths than those of the exposed areas (Fig. 4.1.1b, e). The ridge regions of both species display a similar brownish color and are rarely covered by the transparent surface layer (Fig. 4.1.1c, f). A distinct polygonal surface

- 32 -

Chapter 4 | Multilayer Structures (Ground Beetles) structure (mostly hexagons with a diameter of about 10µm) is present on both the color and ridge regions of both species. However, the hexagonal pattern on the elytra of C. auratus seems to be more pronounced than in C. auronitens (Fig. 4.1.1b, e).

4.2.2. Cross-sectional surfaces of fractured samples The elytra of the two species (60 ~ 200µm in thickness) consist of a ~ 30µm thick dorsal and a ventral, ~15µm thick cuticle region which are partially connected, leaving longitudinally oriented tubular cavities between them. This study focuses on the dorsal cuticle region which is further simply referred to as elytral cuticle. Electron micrographs of cross-fractured elytral cuticle from C. auronitens and C. auratus show that it consists of a dense part close to the surface and an underlying part with twisted plywood structure typical for arthropod procuticle (Fig. 4.2.1a and Fig. 4.2.2a). The distal part can be further subdivided into two structurally distinct layers in both the color (Fig. 4.2.1b and Fig. 4.2.2b) and ridge regions (Fig. 4.2.1c and Fig. 4.2.2c) of both species. The proximal layer appears densely stratified in both investigated regions and does not differ significantly between the two species. In the color regions of both beetles, the distal layer is also stratified with the sub-layers being slightly thicker than those of the underlying proximal layer (Fig. 4.2.1b and Fig. 4.2.2b). In the ridge regions of both beetles, the distal layer appears to be dense and homogeneous (Fig. 4.2.1c and Fig. 4.2.2c). The distal layer has a thickness between 1 and 2 µm in all probed locations in both beetle species. High magnification scanning electron microcopy (SEM) images of the color regions of C. auronitens and C. auratus reveal that individual sub-layers inside the distal layer consist of two types of layers with different thicknesses which are arranged periodically (Fig. 4.2.1d and Fig. 4.2.2d, the thinner layers are indicated by arrows).

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Structural characterization

Figure 4.2.1: SEM images of cross-fractured elytral cuticle of C. auronitens. (a) Overview of the cuticle in color region. The arrows indicate the rotation of twisted plywood structure (ND: normal direction). (b, c) Distal parts of the cuticle in the color and ridge region, respectively (dashed lines: the border between the distal layer and the densely stratified proximal layer). (d) High resolution micrographs of the distal layer in the color region. The arrows indicate the thinner layers.

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Chapter 4 | Multilayer Structures (Ground Beetles)

Figure 4.2.2: SEM images of cross-fractured elytral cuticle of C. auratus. (a) Overview of the cuticle in color region. The arrows indicate the rotation of twisted plywood structure (ND: normal direction). (b, c) Distal parts of the cuticle in the color and ridge region, respectively (dashed lines: the border between the distal layer and the densely stratified proximal layer). (d) High resolution micrographs of the distal layer in the color region. The arrows indicate the thinner layers.

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Structural characterization

4.2.3. Cross-sectional surfaces of microtomed samples Cross-sections of elytral cuticle from both species polished using an ultra-microtome were used to measure and characterize the microstructure of the distal layer. In the color regions of both species, the distal layer is presented as a multilayer structure consisting of bi- layers formed by periodically alternating light and dark contrasted layers (Fig. 4.2.3a, b). There are seven and eight to ten such bi-layers in the multilayer structure of C. auronitens (Fig. 4.2.3a) and C. auratus (Fig. 4.2.3b), respectively. The dark contrasted layer in each bi-layer is thinner than the light contrasted layer. Outwards, the distal layer is completed by an outermost layer which is light contrasted, but is about 30 nm thinner than the light contrasted layers underneath. The thicknesses of the bi-layers, light contrasted layers, dark contrasted layers and the outermost layers of both species are almost equal (Table 4.2.1). In the ridge regions of both species, the distal layer does not form a multilayer structure. Here, it consists of only two parts: a proximal layer with irregular structure which measures about two thirds of the total thickness and a close to surface layer which appears homogeneous and light contrasted in cross section (Fig. 4.2.3c, shown for C. auronitens). In both color and ridge region, a densely stratified layer is presented under the distal layer. The thicknesses of these layers are 64±7 nm and 62±9 nm in C. auronitens and C. auratus, respectively.

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Chapter 4 | Multilayer Structures (Ground Beetles)

Figure 4.2.3: Microstructure of the distal layer and the underlying densely stratified layer in the ultra-microtome polished cross sectional surfaces of elytral cuticle. (a) Color region of C. auronitens and (b) color region of C. auratus showing a multilayer structure. (c) Ridge region of C. auronitens, note that the multilayer structure is absent from the distal layer in this region.

Table 4.2.1: Structural parameters of the multilayer structure in the color regions of the elytral cuticle of C. auronitens and C. auratus.

Average thickness (nm) Number Color of bi- Bi- Distal Dark Light region layers Outermost layer layer contrasted contrasted (N) layer (t) (a=(D- (D) layer (d ) layer (d ) 1 2 t)/N) 7 1195±21 88±5 20±3 137±12 158±3 C. 7 1157±12 102±5 18±3 132±9 151±2 auronitens 7 1186±10 112±5 22±5 127±13 153±1 Average thickness of all samples 101±11 20±4 132±12 154±4 8 1460±39 97±7 24±10 141±18 170±5 C. auratus 8 1369±21 96±3 22±6 129±17 159±3 10 1587±9 98±4 22±6 121±16 149±1 Average thickness of all samples 97±5 22±7 130±18 159±9

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Structural characterization

4.2.4. Oblique sections of microtomed samples When the elytra are microtome polished parallel to the surface, the cuticle is exposed at an oblique angle with respect to the surface normal due to the inherent curvature and roughness of the cuticle. The resulting surface of the cuticle of C. auronitens shows three different layers (Fig. 4.2.4). The optical micrograph (Fig. 4.2.4a) shows that the top layer (layer A, close to surface) is a color generating layer which consists of about 8 sub-layers. From the SEM image of this layer, it is difficult to distinguish the boundaries between the adjacent top three sub-layers (Fig. 4.2.4b, c), although they are visible in the optical micrograph (Fig. 4.2.4a). The underlying five sub-layers were delaminated after polishing, thus show clear boundaries in between (Fig. 4.2.4b, c). Proximally, the layer A is followed by a dark brownish colored layer B (Fig. 4.2.4a), where the observed sub-layers are thinner than those in the layer A (Fig. 4.2.4c), resulting in a finer lamination. Below layer B, a lighter brownish colored layer C can be observed (Fig. 4.2.4a), which consists of planes formed by bundles of parallel fibers with much larger interval distances than those in the layers A and B (Fig. 4.2.4b, c). In case the surface was exposed at a highly oblique angle, large areas of single sub-layers in the color generating layer are directly visible under OM (Fig. 4.2.5a). The most proximal sub-layer (layer 7 in Fig. 4.2.5a) at the bottom of the color generating layer shows a brownish color (originating from the underlying layer) interspersed with some bluish regions. The coloration of the sub-layers changes to bluish green and becomes more and more intense from the proximal to the distal outermost sub-layers (from layer 7 to layer 1 in Fig. 4.2.5a). These areas correspond to the light contrasted sub-layers in the multilayer structure observed in polished cross-sections of the distal layer (Fig. 4.2.3). The coloration at the border between two bluish green sub-layers is always dark blue with a sudden change to orange.

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Chapter 4 | Multilayer Structures (Ground Beetles)

Figure 4.2.4: Optical (a) and electron (b) micrographs showing the microstructure of the same color region in the cuticle of C. auronitens exposed by oblique cutting. (c) High resolution SEM image of layers A and B in (b).

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Structural characterization

Occasionally broad regions showing orange color were exposed (Fig. 4.2.5b). Although it is very difficult to find the boundary between the bluish green and orange regions from the SEM image, these two regions of the same area are optically very distinct confirming the existence of two types of sub-layers (Fig. 4.2.5c). The width of the orange region is normally much smaller than the one of the bluish green region, which implies that this orange region may belong to the thinner dark contrasted sub-layers in the multilayer structure (Fig. 4.2.3).

Figure 4.2.5: Optical (a, b) and electron (c) micrographs showing the microstructure of the color generating multilayer structure in the cuticle of C. auronitens exposed by oblique cutting through a slightly bulged sample. Layers numbered from 1 to 7 indicate the sub-layers (light-contrasted layers observed in polished cross-sections) from distal to proximal. The double headed arrows in (b) and (c) indicate the span of the same bi-layer observed with different microscopy techniques. The obliquely polished surfaces of the cuticle of C. auratus (Fig. 4.2.6a) show a similar outer color generating layer consisting of about 8 sub-layers like the one of C. auronitens (Fig. 4.2.5a). SEM images of this layer show that each individual sub-layer consists of two alternating layers with different microstructure: an outer one whose

- 40 -

Chapter 4 | Multilayer Structures (Ground Beetles) sectioned surface is decorated with dark contrasted dots inside and inner one with a homogenous appearance (Fig. 4.2.6b). In contrast to the sub-layer structure in the outer color generating layer of the cuticle of both species, no such structure is observed (Fig. 4.2.7b) in the brownish outer layer in the ridge region of the elytral cuticle of C. auronitens (Fig. 4.2.7a).

Figure 4.2.6: Optical (a) and electron (b) micrographs showing the microstructure of the color generating multilayer structure in cuticle of C. auratus exposed by oblique cutting.

Figure 4.2.7: Optical (a) and electron (b) micrographs showing the microstructure of the cuticle of C. auronitens in the ridge region exposed by oblique cutting.

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Structural characterization

4.2.5. Structure and color changes of NaOH treated samples Exposure to aqueous NaOH solutions is a well-established method to selectively remove proteins from biological materials (Khor, 2001). A very short wash with NaOH (1.5 M, 8 min.) already led to a dramatic change of the visible color from green (Fig. 4.2.8a) to red (Fig. 4.2.8b) in the color region of C. auronitens, but C. auratus elytra showed almost no change of color after this treatment (Fig. 4.2.8c, d). A similar color change to red occurred for C. auratus however after longer exposure (90 min., Fig. 4.2.8e).

Figure 4.2.8: Color change of elytral cuticle after exposure to aqueous NaOH (1.5M) solution. Color region of C. auronitens before (a) and after (b) 8 minutes NaOH treatment, a change from green to red is observed. Almost no change of color occurred in the color region of C. auratus before (c) and after (d) 8 minutes NaOH treatment, the change to red occurs after 90 minutes (e). After 1 day of NaOH (1M) treatment, the color region of the elytra of C. auronitens has changed back to a green closely resembling the original color (Fig. 4.2.9a). After longer exposure (3 days), this color blue-shifted to purple (Fig. 4.2.9b). After even longer exposure (7 and 10 days), the cuticle displays tan (Fig. 4.2.9c) or orange (Fig. 4.2.9d) colors. The original polygonal surface structure is interrupted by wrinkles and became obscure after 1 day (Fig. 4.2.9a), which

- 42 -

Chapter 4 | Multilayer Structures (Ground Beetles) eventually disappeared after longer exposure (Fig. 4.2.9b-d). SEM images of the cross-sectional surfaces of the multilayer structure in the distal layer show that the number of bi-layers is unaffected by NaOH treatment, but the thicknesses of the sub-layers inside have changed (Fig. 4.2.9e-h). Compared to the untreated cuticle, after 1 day, the thicknesses of the dark contrasted sub-layers have increased to about 74 nm and those of the light contrasted sub-layers have decreased to about 102 nm (Fig. 4.2.9e). After three days, the dark contrasted sub- layers became much thinner than the light contrasted ones (Fig. 4.2.9f). This appearance and the overall thickness of the distal layer (about 900 nm) are largely unchanged after longer exposure (Fig. 4.2.9g, h). The thicknesses of the sub-layers are about 90 nm and 17 nm for light and dark contrasted layers after ten days, respectively (Fig. 4.2.9h). After exposed to NaOH solution (1 M) for 1 day, the color region of the elytra of C. auratus turned from red (Fig. 4.2.8e) back to a blue- green (Fig. 4.2.10a). After 3 days, the color turned to purplish blue interspersed with some areas of green hue (Fig. 4.2.10b). With longer exposure, the color changes to rose after 7 days (Fig. 4.2.10c) and finally to tan after 10 days (Fig. 4.2.10d). On the surface, an onset of wrinkles arising at the boundaries of the hexagons is observed after 1 day (Fig. 4.2.10a). These wrinkles became pronounced after 3 days (Fig. 4.2.10b) and converged to longer ones with irregular distributions after 7 and 10 days (Fig. 4.2.10c, d). The polygonal structures on the surface were obscured by the wrinkles, but still recognizable after 10 days (Fig. 4.2.10d). Microstructural changes of the distal layer after 1 day and 3 days cannot be determined precisely, since in all probed samples its cross-sectional surfaces was irregularly covered with partially grainy material (Fig. 4.2.10e, f). After 7 days, a decrease in thickness of both the light and dark contrasted sub-layers is observed (Fig. 4.2.10g), while a further decrease after 10 days is not noticeable (Fig. 4.2.10h).

- 43 -

Structural characterization

Figure 4.2.9: Effects of exposure to NaOH (1 M) for different times on color, surface (a-d, OM images) and the microstructure of the multilayer structure (e-h, SEM images of cross-sectional surfaces) in elytral cuticle of C. auronitens. (a, e) 1 day, (b, f) 3 days, (c, g) 7 days, (d, h) 10 days.

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Chapter 4 | Multilayer Structures (Ground Beetles)

Figure 4.2.10: Effects of exposure to NaOH (1 M) for different times on color, surface (a-d, OM images) and the microstructure of the multilayer structure (e-h, SEM images of cross-sectional surfaces) in elytral cuticle of C. auratus. (a, e) 1 day, (b, f) 3 days, (c, g) 7 days, (d, h) 10 days.

- 45 -

Structural characterization

The NaOH treatment also revealed some structural features on the surfaces of the elytra of both species. After exposed to NaOH (1 M) for 1 day, small flakes embedded in the outermost layer of the color region of C. auronitens were observed (right part of Fig. 4.2.11a). No similar structures were observed in the underlying layer (left part of Fig. 4.2.11a). In the transition zone from the color to ridge region, densely packed lens-shaped particles were observed (Fig. 4.2.11b). After 7 days, all these particles or flakes disappeared and cleaner surfaces were observed on both the color (Fig. 4.2.11c) and ridge region (Fig. 4.2.11d).

Figure 4.2.11: SEM images of the ultrastructural changes on the surfaces of elytra from C. auronitens after exposure to NaOH (1M) for different times. (a) Small flakes appear in the outermost layer of the color regions (1 day). (b) Densely packed lens-shaped particles in transition zone from the color to ridge region (1 day). Clean surfaces appear in both color region (c) and ridge region (d) after exposure for 7 days. In the case of C. auratus, after exposure to NaOH (1.5 M) for 1.5 hours, some lens-shaped particles appeared on the surface of the color regions (Fig. 4.2.12a). After longer exposure to NaOH (1 M) for 7 days, another special flake like structure was revealed (Fig. 4.2.12b).

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Chapter 4 | Multilayer Structures (Ground Beetles)

These flakes are arranged in individual domains where they are all oriented to the same direction (arrows in Fig. 4.2.12b). Some areas of the outermost layers were occasionally removed showing inner smooth surfaces of a terrace like structure which can come from the possible broken multilayer structures (indicated by arrows in Fig. 4.2.12c). The flakes shown in the lower right corner of Fig. 4.2.12c is lying above these multilayer structures. After 10 days, most areas of these structures were washed away with a few flakes left (indicated by arrows in Fig. 4.2.12d).

Figure 4.2.12: SEM images of the ultrastructural changes on the surfaces of elytra from C. auratus after exposure to NaOH for different times. (a) Lens- shaped particles appear in the outermost layer of the color regions (1.5 M NaOH, 1.5 hours) (b) Oriented flake like particles arranged in domains (arrows indicate the orientations) appear after 7 days (1 M NaOH). (c) Superimposed thin layers (arrows) under the flakes in areas where the multilayer structure broke off (1 M NaOH, 7 days). (d) Residual flakes (arrows) after exposure for 10 days (1 M NaOH).

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Experimental investigation of the optical properties

4.3. Experimental investigation of the optical properties

4.3.1. Reflectance at normal incidence The reflectivity spectra of the elytra of both species at normal incidence were measured in an OM using objectives of different magnifications. For objectives of 20 × and 50 ×, maximum reflectivity peaks locate at 553 nm and 550 nm for C. auronitens, respectively (Fig. 4.3.1a). For C. auratus, these peaks locate at longer wavelengths of 574 nm and 573 nm, respectively (Fig. 4.3.1b). For an objective of 100 ×, the peaks of both species blue-shift to 538 nm and 565 nm for C. auronitens (Fig. 4.3.1a) and C. auratus (Fig. 4.3.1b), respectively. The reflectivity of both species measured at a higher magnification is higher than that measured at a lower magnification (Fig. 4.3.1). The reflectivity of C. auronitens at 10 ×and 5 × magnifications (Fig. 4.3.1a) and C. auratus at 5 × (Fig. 4.3.1b) is so low that it is difficult to determine the position of peaks. For C. auratus at 10 × magnification, the peak locates at 576 nm (Fig. 4.3.1b), which is very close to the positions of peaks at higher magnifications of 20 × and 50 ×. From the measured spectra at normal incidence, one can calculate the average refractive index of the distal layer using the equation = /2 (see Section 2.3.2), where λ is the wavelength of maximum reflectivity peaks (Fig. 4.3.1)푛� and a is the thickness of the bi-layers (Table푛� 휆 4.2.1).푎 The calculated is 1.80 and 1.81 for C. auronitens and C. auratus, respectively. 푛�

Figure 4.3.1: Reflectivity spectra of the elytra of C. auronitens (a) and C. auratus (b) at normal incidence measured in an OM using objectives of different magnifications indicated by differently colored curves. The wavelengths of maximum reflectivity are indicated on the graphs. - 48 -

Chapter 4 | Multilayer Structures (Ground Beetles)

4.3.2. Reflectance at different angles of incidence The reflectivity spectra of both species at incidence angles of 50°, 60° and 70° for both s and p polarizations are shown in Fig. 4.3.2. The maximum reflectivity of both species at these oblique incidence angles is very low, less than 3 %. The reflectivity of p polarization is even lower (less than 0.5 %) than that of the s polarization. Therefore, the peak positions of the s polarization are easier to be determined than those of the p polarization and thus the s polarization peaks were used for optical analysis. These peaks locate in the green to blue region of the spectra, which is coinciding with the observation at an oblique angle over the elytra of both beetles. The peaks gradually shift to shorter wavelengths when the angles of incidence increase from 50° to 70°. The wavelengths of all peaks of C. auratus are longer than those of C. auronitens at the same angle of incidence.

Given the different wavelengths of maximum reflectivity ( , ) determined at different angles of incidence ( , ) as shown in Fig. 1 2 4.3.2., one can calculate the average refractive indices of the휆 distal휆 1 2 layers of both beetles using Eq. 4.3.1: 휃 휃 푛� sin sin = (4.3.1) 2 2 2 2 휆1 휃2 − 휆2 휃1 derived from Eq. 2.3.2.푛� �The calculated2 2 of the distal layers for C. 휆1 − 휆2 auronitens and C. auratus are 1.70 and 1.77, respectively. 푛�

Figure 4.3.2: Reflectivity spectra of the light in an angular interval of ~2° around the specularly reflected beam at incidence angles of 50°, 60° and 70° for C. auronitens (a) and C. auratus (b). (ss and pp: s polarization and p polarization, respectively, for both polarizer and analyzer).

- 49 -

Experimental investigation of the optical properties

The angular dependence (25° - 85°) of reflectivity of both species for both s and p polarizations at 532 nm are shown in Fig. 4.3.3. For C. auronitens (Fig. 4.3.3a), the reflectivity of p polarization is always lower than the one of s polarization. In contrast, for C. auratus (Fig. 4.3.3b), the reflectivity of p polarization is higher than the one of s polarization in the angular ranges of 25° - 46° and 74° - 85°. In general, the reflectivity of s polarization increases with the increment of the angle of incidence for both species, whereas the reflectivity of p polarization has a minimum value at about 58.4° for C. auronitens (Fig. 4.3.3a) and 60.7° for C. auratus (Fig. 4.3.3b), i.e. the Brewster’s angles ( ) for the distal layers of both species at 532 nm. Given these two angles, one can calculate the average refractive indices of the 퐵 distal layers휃 of both species according to the Brewster’s law tan = (the refractive index of air = 1). The calculated 푛� is 1.63 퐵 for C. auronitens and 1.78 for C. auratus. 휃 푛�⁄푛푎푖푟 푛푎푖푟 푛�

Figure 4.3.3: Angular dependence of reflectivities at λ=532 nm from the elytra of C. auronitens (a) and C. auratus (b). (ss and pp: s polarization and p polarization, respectively, for both polarizer and analyzer). Reflected intensity was collected in an angular interval of ~ 2° around the specularly reflected beam.

4.3.3. Scattering at different angles of incidence The angular dependence of the intensity of incidence light (λ = 532 nm) which is scattered from the elytra of both species at three different angles of incidence (30°, 50° and 70°) is shown in Fig. 4.3.4. The reflected light spreads over a large range of angles around specular reflection directions. For p polarization at 50°, the scattered

- 50 -

Chapter 4 | Multilayer Structures (Ground Beetles) light is even stronger at lower angle of detection than those at the angle around specular reflection (green curves in Fig. 4.3.4b, d).

Figure 4.3.4: Angular dependence of the intensity of light (Iscat, λ=532nm) scattered from the elytra of C. auronitens (a, b) and C. auratus (c, d) at different angle of incidence (θi) as illustrated by the vertical dashed lines. (I0: the intensity of incident light, θscat: the angle of scattered light, ss and pp: s polarization and p polarization, respectively, for both polarizer and analyzer).

4.4. Simulation of the optical properties

4.4.1. Reflectance at normal incidence In order to systematically study the influence of the structural parameters of the biological one-dimensional (1D) photonic crystals on their optical properties, I calculated the reflectance spectra of a model based on the morphological values obtained from the biological structure (inset in Fig. 4.4.1), and also with artificial parameters that

- 51 -

Simulation of the optical properties slightly differ from those of the biological model. This model mainly consists of a multilayer structure formed by two types of sub-layers with different refractive indices (1.56 and 2.00 for the light and dark contrasted layers, respectively) and different thicknesses (see Table 4.2.1). The top and bottom layers of the multilayer structure are both dark contrasted layers. Above the top dark contrasted layer is the outermost layer (see thickness in Table 4.2.1), whereas under the bottom dark contrasted layer is a 10 µm thick substrate layer. Both the outermost layer and the substrate layer are assumed to have a refractive index of 1.56, the same as the one of the light contrasted layer. The incidence medium is air with a refractive index of 1.00. The calculated reflectance spectra at normal incidence show broad peaks with maximum reflectivity at 506 nm and 503 nm for C. auronitens and C. auratus, respectively (gray curves in Fig. 4.4.1.). The measured spectra (black curves in Fig. 4.4.1) show peaks in a longer wavelength region compared to the simulation results. The calculated maximum reflectivity of C. auratus (58 %) is higher than the one of C. auronitens (42 %). The measured spectra of both species show lower reflectivity than those of the calculated ones, and C. auronitens shows higher reflectivity than the one of C. auratus.

Figure 4.4.1: Calculated reflectivity spectra at normal incidence for C. auronitens (-■- curve) and C. auratus (-▲- curve) based on the inset structural model (1: outermost layer, 2: dark contrasted layer, 3: light contrasted layer, 4: substrate layer, where the structural parameters of layers 1-3 for both species were taken from Table 4.2.1 and the thickness of layer 4 is 10 µm). The -■- and -▲- curves show the measured spectra of C. auronitens and C. auratus, respectively (see also Fig. 4.3.1), for comparison.

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Chapter 4 | Multilayer Structures (Ground Beetles)

4.4.2. Influence of different angles of incidence The calculated reflectance spectra at non-normal incidence (Fig. 4.4.2) show that for both s (solid curves) and p polarizations (dashed curves) the reflection peaks shift to shorter wavelengths when the angle of incidence increases from 50° to 70°. The experimentally measured positions of reflection peaks of s polarization (vertical lines) are located at longer wavelength regions than those of the simulation results. The calculated maximum reflectivity of both polarizations is higher than the experimentally determined reflectivity (Fig. 4.3.2).

Figure 4.4.2: Calculated reflectivity spectra of C. auronitens (a) and C. auratus (b) based on the multilayer model in Fig.4.4.1. The experimentally measured positions of reflection peaks (see also Fig. 4.3.2) are marked as vertical lines in both figures. (ss and pp: s polarization and p polarization, respectively, for both polarizer and analyzer).

4.4.3. Influence of the number of bi-layers When the number of the bi-layers in the model structure varies from the number of bi-layers formed by the beetles but the other structural parameters are kept the same, the position of the reflectance peaks of both species does not move, but the maximum reflectivity changes (Fig. 4.4.3). For C. auronitens, the maximum reflectivity increases to 50 % when one more bi-layer (8 bi-layers) is included in the model (black dashed curve) and decreases to 34 % when one bi-layer (6 bi- layers) is removed from the model (black dotted curve). For C. auratus, one bi-layer more or less than the number of bi-layers formed by the beetle increases or decreases the maximum reflectivity to 65 % (red dashed curves) or 48 % (red dotted curves), respectively.

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Simulation of the optical properties

Figure 4.4.3: Calculated reflectivity spectra at normal incidence for C. auronitens (black curves) and C. auratus (red curves) based on the modified multilayer model in Fig. 4.4.1 by adding (dashed curves) or removing (dotted curves) one bi-layer to or from the original model (solid curves).

4.4.4. Influence of varying thicknesses of individual layers The multilayer model in Fig. 4.4.1 with constant thickness for individual layers is an idealized model of the observed multilayer structure in the cuticle where the layer thicknesses normally vary in the vicinity of an average value. In order to check whether this idealized model can reproduce the optical characteristics of the real structure, I compare the reflectivity spectra of the idealized model and a modified model by varying the thicknesses of the individual layers from the average values measured from the biological structure (Table 4.2.1) within the range of the determined standard deviation. The overall average thickness values for individual layers remain unchanged though. The spectra calculated for both species on this modified model with varying layer thicknesses (solid curves in Fig. 4.4.4) show the similar reflectivity (difference < 5 %) and reflection peaks of close positions (difference of ±16 nm) to those calculated on the model with constant layer thicknesses (dashed curves in Fig. 4.4.4).

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Chapter 4 | Multilayer Structures (Ground Beetles)

Figure 4.4.4: Calculated reflectivity spectra at normal incidence for C. auronitens (a) and C. auratus (b) based on a multilayer model with varying layer thicknesses (solid curves) modified from the idealized model in Fig. 4.4.1 with constant layer thicknesses (dashed curves). The variations of the thickness from the average thickness of individual layers are within the bounds of the standard deviation of the measurements (see Table 4.2.1).

4.4.5. Influence of the thickness of the outermost layer Since I found that the thicknesses of the outermost layer of the biological 1D photonic crystals in both species have different values compared to the light contrasted layers in the underlying multilayer structure, I calculated the reflectance spectra at normal incidence dependent on the thickness of the outermost layer (Fig. 4.4.5a and Fig. 4.4.6a) to see its influence on the optical properties of the photonic structure. The reflectance spectra of the model with thinner (20 nm less than the original one, dashed curves) or thicker (as thick as the underlying light contrasted layer, dotted curves) outermost layers show higher maximum reflectivity and narrower peak widths compared to those of the model with the outermost layers of the original thickness formed by the beetles (solid curves).

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Simulation of the optical properties

Figure 4.4.5: (a) Calculated reflectivity spectra at normal incidence for C. auronitens based on the modified model in Fig. 4.4.1 by varying the thickness of the outermost layer (dashed and dotted curves) and comparison to the calculated spectrum of the original model (solid curve). (b) FWHM (-■- curve) and maximum reflectivity (-▲- curve) of spectra in (a) in the function of the thickness of the outermost layer. (vertical dotted line: the thickness of the outermost layer formed by C. auronitens).

Figure 4.4.6: The same as Fig. 4.4.5 but for C. auratus. Simulation results show that the thicknesses of the outermost layers formed by both C. auronitens (Fig. 4.4.5b) and C. auratus (Fig. 4.4.6b) are the minimum thickness to obtain the broadest full width at half maximum (FWHM) of the reflection peaks (-■- curves) and the lowest maximum reflectivity of the spectra (-▲- curves). For C. auronitens, between the model with the outermost layer of the original thickness and the model with the outermost layer as thick as the one of the underlying light contrasted layer, the differences of the FWHM and maximum reflectivity are 36 % and 40 %, respectively. For C.

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Chapter 4 | Multilayer Structures (Ground Beetles) auratus, these differences are 24 % and 21 % for the FWHM and maximum reflectivity, respectively.

4.4.6. Influence of the thickness of the transparent surface layer In both species, but especially in C. auronitens, large areas of the elytra are covered by a transparent surface layer (see Fig. 4.1.1b) located above the outermost layer which corresponds to the wax layer described in other insects. The thickness of this layer varies from about 50 nm to 500 nm in different regions of the elytra of C. auronitens. The refractive index of this transparent surface layer was assumed to be 1.40 which is the value determined from the cuticular wax on the wing of a dragonfly species at 543 nm (Hooper et al., 2006). The calculated spectra at normal incidence for wax layers with different thicknesses are shown in Fig.4.4.7. The peak positions of most spectra of the models with a wax layer (solid curves) shift to a shorter wavelength region compared to that of the model without wax layer (dashed curves) (Table 4.4.1). The reflectivity of models with wax layers is higher than the one without wax layer. The highest maximum reflectivity is obtained when the thickness of the wax layer (100 nm) is almost equal to the thickness of the outermost layer (Table 4.4.1).

Figure 4.4.7: Calculated reflectivity spectra at normal incidence for C. auronitens based on the modified multilayer model in Fig. 4.4.1 by adding one wax layer on top of the outermost layer and varying its thickness (solid curves) comparing to the spectra calculated on the original model without wax layer (dashed curve).

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Discussion

Table 4.4.1: Peak positions and maximum reflectivity of the spectra calculated for C. auronitens (see Fig. 4.4.7) in dependence of the thickness of the wax layer.

Thickness of wax 0 50 100 200 300 400 500 layer (nm) Peak position (nm) 506 481 494 475 501 481 508 Maximum 42 % 59 % 63 % 53 % 61 % 60 % 58 % reflectivity

4.5. Discussion

4.5.1. Photonic structures of the Ground beetle species For both fractured samples (Fig. 4.2.1d and Fig. 4.2.2d) and cross- sectionally microtomed samples (Fig. 4.2.3a, b) from both species, I observed multilayer structures forming the distal layers of the cuticles in the color regions of the elytra. The optical micrographs of the obliquely microtomed samples directly show that the color generating top layer is formed by the multilayer structures (Fig. 4.2.4a, Fig. 4.2.5a and Fig. 4.2.6a). In contrast, such structures are absent from the distal layers of the cuticle in the ridge regions (Fig. 4.2.1c, Fig. 4.2.2c, Fig. 4.2.3c and Fig. 4.2.7). Therefore these unique multilayer structures are the primary origin of the distinct greenish color in the color regions compared to the dark brownish appearances of the ridge regions. Regarding the origin of similar multilayer structure like the one found in this study, there are two possible assignments under discussion. Some studies described this structure in beetle cuticles as exocuticle (Vigneron et al., 2006; Noyes et al., 2007). According to this hypothesis, the multilayer structure has been considered to be chitin based due to the chitinous nature of exocuticle. Other authors have described this structure as a part of the epicuticle (Schultz and Rankin, 1985b; Kurachi et al., 2002). The results of this study support this second hypothesis. The epicuticle is known to be the outermost layer in the exoskeleton of Arthropoda (Filshie, 1982, see also Section 2.2). The location of the multilayer structure I observed is, apart from the superposed transparent surface layer, always the outermost

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Chapter 4 | Multilayer Structures (Ground Beetles) structure within the elytral cuticle (Fig. 4.2.1d, Fig. 4.2.2d, Fig.4.2.3a, b, Fig. 4.2.4a and Fig. 4.2.6a). In addition, I observed pronounced reductions of the thickness of the multilayer structure after exposure to NaOH for more than three days (Fig. 4.2.9 f-h and Fig. 4.2.10g, h). These decreases were not only directly proved by measuring the thickness of the multilayer structures, but are also supported by the change of the elytral color to shorter wavelengths during the ongoing NaOH treatment (compare Fig. 4.2.9a to b, Fig. 4.2.10a to b), since both mathematical methods (see Section 2.3.2) and empirical experience show that thinner layers result in shorter wavelengths of reflected light (Lenau and Barfoed, 2008). The red-shift of the color of the cuticle (from green to red) after exposure to NaOH solution for less than 1 day (Fig. 4.2.8) is most probably a result of initial swelling of the multilayer structure, which increases the thickness of the layers and hence the wavelength of the reflected light. This swelling is still visible after 1 day of exposure indicated by the increment of the thickness of the dark contrasted layer from original 20 nm to 74 nm (Fig. 4.2.9e). However, despite of this increase in thickness, the reflected color does not differ too much from the untreated one (Fig. 4.2.9a). It is because of both the reduction of the refractive index of this sub-layer, caused by the substitution of the biological material by water with low refractive index, and the reduction of the thicknesses of the light contrasted layers. The reduction of the thickness of the multilayer structure is most probably caused by denaturing and removal of biological molecules, like proteins and lipids, as a result of the reaction of NaOH and cuticle. The NaOH solution used in this study (1 M and 1.5 M) cannot dissolve chitin. If the multilayer structure would belong to the chitin based exocuticle, the NaOH treatment should have left over fibrillar structures arranged in a twisted plywood fashion as described to be typical for exocuticle (Hadley, 1986). However, this was not the case here. All these results indicate that the multilayer structure is a part of the epicuticle and chemically distinct from the chitinous layers below. Underlying the multilayer structure there is always an even thinner layered structure (densely stratified layers in Fig. 4.2.3 and layer B in Fig. 4.2.4) which is bordered by the typical twisted plywood structure associated with endocuticle (Fig. 4.2.1a and Fig. 4.2.2a and layer C in Fig. 4.2.4).

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Discussion

Consequently, it is safe to assume that the thin layers under the multilayer structure constitute the exocuticle. It is known that epicuticle may be subdivided into two layers: outer epicuticle and inner epicuticle (Weis-Fogh, 1970; Wigglesworth, 1975, see also Section 2.2). The inner epicuticle, whose thickness ranges from 0.5 to 2µm, forms the bulk of the epicuticle of insects (Neville, 1975; Wigglesworth, 1985). The multilayer structure found here also forms the major part of the epicuticle of the investigated two beetles (Fig. 4.2.3a, b). The thickness of this structure is between 1 and 1.5 µm (Table 4.2.1), which is similar to the thickness of the inner epicuticle of other insect species. Therefore, one can assume that the multilayer structure is formed by the inner epicuticle in both beetles. For the investigation of the optical properties of the multilayer structure, it is necessary to know the refractive indices of the constituting light and dark contrasted sub-layers, which requires the knowledge of the chemical composition of both layer types. In the hardened cuticle of adult beetles, the sclerotized structural lipid is the main component of the inner epicuticle (see Section 2.2). The chemical treatment of the cuticle of the two investigated Ground beetle species shows that the light contrasted layers of the multilayer structure are still present after exposure to NaOH for 10 days (Fig. 4.2.9h and Fig. 4.2.10h), which indicates their strong resistance to chemical degradation. Therefore, I speculate that this layer is mainly composed of structural lipids, which is known to be very resistant to chemical degradation (see Section 2.2). In contrast, during the same treatment, the dark contrasted layers are not as chemically stable as the light contrasted ones, since they almost disappeared after 10 days exposure to NaOH (Fig. 4.2.9h and Fig. 4.2.10h). Schultz and Rankin (1985b) studied similar multilayer structures in the elytral cuticle of tiger beetles. The authors proposed that the electron-dense layer in the multilayer structure maybe consists of melanin or melanoprotein, which provides the unusually high refractive index of 2.0 for this layer. This electron-dense layer in their investigation by TEM shows similar higher solubility in alkali solution than the electron-lucent layer as in this study the dark contrasted layer does compared to the light contrasted layer. This implies that the dark contrasted layers observed here are also mainly composed of melanin and proteins.

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Chapter 4 | Multilayer Structures (Ground Beetles)

During the darkening and hardening of insect cuticle, both, melanin responsible for coloration, and, quinones responsible for sclerotization originate from dopamine by different biosynthesis pathways. Evidence has been shown that if more dopamine was diverted into the melanization pathway, the resulting cuticle properties are weaker because of deficiencies of tanning agents (Hopkins and Kramer, 1992). That means the proteins inside the dark contrasted layers are less sclerotized than those inside the light contrasted layers. Consequently, the material inside the dark contrasted layers has higher solubility in the NaOH solution than the one in the light contrasted layers. Therefore, a melanin nature of the dark contrasted layers could explain their lower stability towards the alkali treatment compared to the light contrasted layers. Another observation indicating the occurrence of melanin in the dark contrasted layer is that these individual thinner layers in the multilayer structure exposed by oblique microtome polishing display a reddish color (Fig. 4.2.5a, b) which is typical for phaeomelanin (Riley, 1997). Although the exact chemical composition or refractive indices of the two types of layers in the multilayer structure could not be determined here, the results in this study indicate that structural lipids and melanin are probably the appropriate candidates for the constituents of the light and dark contrasted layer, respectively. It is thus not probable that chitin is one of the materials involved in the formation of the epicuticular reflectors. Although the widely accepted value of 1.56 for the refractive index of the beetle reflectors was originally determined for chitinous material (Vukusic et al., 1999), the building material for these reflectors studied here not necessarily has to be chitin based. However, the recurrence of the value of 1.56 indicates that the refractive index of one type of the layers in the epicuticular reflectors has about the same value, despite its different chemical composition. On top of the multilayer structure, I found one light contrasted layer which is about 30 % thinner than the other light contrasted layers below (Fig. 4.2.3a, b and Table 4.2.1). Due to its outermost position, this distinct layer can be assigned as belonging to the outer epicuticle. However, it is also possible that outer epicuticle is one part of this layer, since this layer is much thicker than the outer epicuticle

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Discussion described in literature. Schultz and Rankin (1985b) described the outer epicuticle of tiger beetles as a 30 to 40nm thick electron dense region with possible ultrastructure inside. Most authors, who are interested in studying structural coloration of epicuticular multilayer reflectors of beetles, normally do not specifically separate the outer epicuticle from the underlying multilayer structure. I found lens-shaped and flake like particles on the top surfaces of the elytral cuticles after NaOH treatment (Fig 4.2.11 and Fig. 4.2.12). For C. auratus, the lens-shaped particles (Fig. 4.2.12a) were revealed on the surface of the color region after exposure to NaOH for 1.5 hours. After 7 days, these particles disappeared and another type of structure, clusters of oriented flakes, was revealed (Fig. 4.2.12b, c). These flakes seem to be dissolved after 10 days (Fig. 4.2.12d). For C. auronitens, small flakes were already observed to be embedded in the outermost layer of the color region after exposure to NaOH for 1 day (Fig. 4.2.11a). However, these flakes do not form clusters like those observed in C. auratus. They are loosely and randomly distributed inside the layer. Another structure, densely packed lens-shaped particles (Fig. 4.2.11b) was revealed on the surface of ridge region after 1 day. All these structures revealed on the elytral surface of C. auronitens were removed after 7 days (Fig. 4.2.11c, d). Since all these particles or flakes were located within the outermost layers of the epicuticle (Fig. 4.2.11a, b and Fig. 4.2.12a, c), they are most probably constituents of the outer epicuticle. The difference of the ultrastructure in the outer epicuticle between the two beetles, namely the occurrence of clusters of oriented flakes, has an influence on the chemical stability of their cuticle. During the NaOH treatment, it took much longer time for the color regions of C. auratus (90 min., Fig. 4.2.8e) to change to the similar reddish color for C. auronitens (8 min., Fig. 4.2.8b) by swelling. This implies that swelling started later in C. auratus than it did in C. auronitens. A possible reason for this difference is that the densely packed flakes act as a permeability barrier which protects the beetle both from desiccating and from the penetration of water from outside into the body via the cuticle. Such structures may help C. auratus, a more thermophilic species than C. auronitens, to better adapt to the more arid environment it lives in. The densely packed flakes of C. auratus is most probably also the reason that the polygonal structures on the

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Chapter 4 | Multilayer Structures (Ground Beetles) surface of its color regions are more stable, still visible even after 10 days of NaOH treatment (Fig. 4.2.10d), than those of C. auronitens which disappeared already after 3 days (Fig. 4.2.9b). The influence of the ultrastructure in the outer epicuticle and the thickness of this outermost layer on the optical properties of the cuticle are discussed in the next section. According to the general model of insect cuticle, the outer epicuticle is covered with two extracuticular layers: the wax layer and the thin cement layer (see Section 2.2). In this study, the transparent surface layer observed on the surfaces of the elytral cuticle of both species (Fig. 4.1.1b, e) can be assumed to be the wax layer, due to its location in the cuticle and the glassy appearance. By means of the characterization methods used in this study, it was not possible to distinguish a cement layer. The superficial wax layer also slightly changes the perceived color of the elytra (Fig. 4.1.1b, e). The influence of the thickness of this layer on the optical properties of the cuticle is discussed in the next section. Based on the results of this study, I propose the following structural model for the color producing epicuticle in the color regions of the elytra of both beetles (Fig. 4.5.1). The top layer is the transparent extracuticular wax layer. The subjacent layer (so-called outermost layer) is formed by or contains the outer epicuticle and also contains different ultrastructures in the two beetles. In C. auratus, this layer contains lens-shaped particles and underneath numerous small flakes that arrange in individual domains where these flakes are all oriented in the same direction. In C. auronitens, the outer epicuticle in color regions may contain loosely and randomly distributed small flakes. The subjacent inner epicuticle contains a multilayer structure consisting of periodically ordered alternating layers with different thicknesses and chemical compositions. The thicker layers (light contrasted layers) are most probably formed by sclerotized structural lipids, whereas the thinner layers (the dark contrasted layers) contain melanin and proteins. This multilayer reflector is the primary origin of the coloration of the cuticle. The number of the bi-layers as well as the thicknesses of individual layers are slightly different in the two beetle species. Wax channels pervading the whole epicuticle as described for other insects were not observed in this study and are therefore not

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Discussion presented in the current model. The inner epicuticle is then followed by the exocuticle with many fine layers and the endocuticle with its twisted plywood structure.

Figure 4.5.1: Model of the epicuticle in the color regions of the elytra of both beetles. The ultrastructure showing in the outer epicuticle is abstracted from those in C. auratus. The ultrastructure inside the outer epicuticle of C. auronitens, which is different from those in C. auratus, is not shown in this model. Note the flakes in the outer epicuticle of C. auratus are more densely packed and ordered than those in C. auronitens. The wax channels are not presented in this model.

4.5.2. Optical properties of the photonic structures The elytra of C. auronitens reflect a yellow-green light, whereas the elytra of C. auratus display a golden color when observed by the eye (Fig. 4.1.1a, d). The difference in wavelengths between the reflectivity maxima of these two beetles measured at normal incidence is around 23 nm (Fig. 4.3.1). In contrast, the calculated spectra of both beetles show two peaks with the maximum reflectivity at almost equal wavelengths (gray lines in Fig. 4.4.1). The overlap of these two peaks is because the measured thicknesses for the light and dark contrasted layers used as inputs in the simulations are almost equal between the two beetles and the same refractive indices were used for both species. In other words, the models for the two beetles are almost identical,

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Chapter 4 | Multilayer Structures (Ground Beetles) except that the volume fraction of the dark contrasted layers in C. auratus is 1 % larger than the one of C. auronitens. This small structural difference leads to a maximum difference of 0.03 in the estimated average refractive indices ( ) of the photonic structure between the two beetles (C. auronitens: 1.59 ≤ ≤ 1.61, C. auratus: 1.59 ≤ ≤ 1.62, see Eq. 2.3.1). This difference푛� in refractive index then leads to the theoretical maximum difference of 푛the� peak positions of about 10푛� nm between the two beetles at normal incidence calculated by Eq. 2.3.3, which is much smaller than the measured one (23 nm, Fig. 4.3.1). In addition to this disagreement, the peak positions of the calculated spectra for both beetles are also different from those of the measured spectra, which are shifted about 50 – 70 nm (Fig. 4.4.1) towards shorter wavelengths compared to the measured ones. This difference is also observed between the calculated and measured spectra at other angles of incidence (Fig. 4.4.2). Under the assumption that the measured thicknesses of the bi-layers in C. auronitens and C. auratus are accurate and similar, these two disagreements between the calculated and measured spectra imply that (i) the average refractive index of C. auratus should be larger than the one of C. auronitens and (ii) the average refractive indices of each of these two beetles should be larger than the theoretically estimated values around 1.61. The average refractive indices derived from three independent optical experiments (Fig.4.3.1-3) support these implications. These experimentally determined values are all larger than the theoretically estimated values. These differences are in the range of 0.02 – 0.2. The smallest difference (0.02) is between the estimated value (1.61) and the one (1.63) derived from the Brewster’s angle of C. auronitens. However this exceptionally small difference occurs most probably because of the difficulty in accurately determining this angle in Fig. 4.3.2a. Between the two species, the experimentally determined average refractive index for C. auratus is always larger than the one for C. auronitens. The smallest difference (0.01) derived from the reflectivity spectra at normal incidence is not as significant as the other two (0.15 and 0.07). However this is because only in this method the measured average thickness of the bi-layers is involved, of which a small variation (5 nm) would also influence the difference of the wavelengths of the reflected light between the two beetles. As a result, the required difference in the average refractive indices to fit

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Discussion the measured spectra can be smaller than the values derived by the other two methods. Therefore, since the possible errors originating from the determination of the critical point of the data (the Brewster’s angle) and the measurement of the structural parameters (the layer thickness) are avoided in the experiment that measures the reflection spectra at different angles of incidence, the average refractive indices derived from these spectra is a more reliable one compared to those from the other two optical experiments. Using the average refractive indices derived by this method, the resulting peak positions of C. auronitens and C. auratus at normal incidence are 524 nm and 563 nm, respectively. These positions agree better with the experimental results than the simulation results do (Fig. 4.4.1). Therefore the average refractive index of the photonic structure in C. auratus is determined to be 1.77 which is larger than the one (1.70) for C. auronitens. This result shows that, even for two closely related species, the variation of the structural color can also originate from the difference in chemical composition of the photonic structure, not only the structural parameters. The absolute values of the derived average refractive indices of both beetles (1.70 and 1.77) are higher than those (1.61 and 1.62) estimated from the model using the widely accepted values 1.56 and 2.00 as inputs for the refractive indices for the light and dark contrasted layers, respectively. If using the refractive indices 1.56 and 2.00 as input values, the required volume fraction of the high index layers (dark contrasted layers) would be at least 31 % in order to achieve the experimentally derived higher average refractive indices. This volume fraction is much higher than the measured one (about 14 %). Therefore, two possible explanations for the difference between the measured average refractive indices and the estimated ones are i) the measured thicknesses of high index layers are smaller than the real values due to the layer shrinkage during SEM observation and ii) the refractive indices of the two sub-layers in the multilayer structures of the two investigated beetles should be different from the widely accepted values. However, the exact values of the refractive indices for individual light and dark contrasted layers of these two species are unknown. So far, the real refractive index (n) of the material has been considered, but the absorption coefficient (k) also plays a role in the coloration of the investigated beetles, since there are strong indications that the dark

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Chapter 4 | Multilayer Structures (Ground Beetles) contrasted layers may contain the absorptive pigment melanin. In literature, there is still little knowledge about the exact chemical composition and local distribution of the materials forming the beetle reflectors. Consequently, there is a general lack of information about their precise refractive indices (Parker et al., 1998). Even for the known constituents, such as chitin and melanin, there is still uncertainty about their real refractive indices and limited knowledge about their absorption coefficients (Shawkey et al., 2009). Noyes et al. (2007) applied an optical characterization method to indirectly quantify the complex refractive indices of these two types of layers of beetle Chrysochroa raja. However the fitting procedure they applied can result in several sets of refractive indices that generate similar spectra (Yoshioka and Kinoshita, 2011). Yoshioka and Kinoshita (2011) directly determined the refractive indices of the individual layers in the multilayer reflector of beetle Chrysochroa fulgidissima through an experimental procedure involving semi-frontal thin- sectioning of the multilayer structure and subsequently optical examinations. The experimental method they applied requires that the thickness of the TEM section to be smaller than the thickness of the individual layers in the multilayer structure, which is difficult to achieve in general sample preparation and especially for the thinner layers of the two investigated ground beetle species. The determined refractive indices for C. raja and C. fulgidissima are all lower than 1.70. In comparison, the average refractive indices of the two ground beetle species (1.70 and 1.77) are relatively higher. The refractive index assumed for the thinner layer (2.00) is even higher. However, it has been shown that synthetic organic semiconductor films can possess refractive index as high as 1.99 (at 532 nm) (Yokoyama et al., 2012). Therefore, it adds the interest in future to investigate the chemical composition of the biological materials formed by these two ground beetle species to reveal what is the strategy of nature to synthesis high refractive index polymers. Besides the color of the light reflected by the cuticle, its intensity, which is defined by the absolute reflectivity of the cuticle, should also play an important role in the ecophysiology of the two investigated beetle species. The maximum reflectivity of the measured spectra at normal incidence (black curves in Fig. 4.4.1) is very small (less than 15 %) and much lower compared to that of the calculated spectra

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Discussion

(gray curves in Fig. 4.4.1). This can be explained by the strong scattering of light to many directions by the biological structure (Fig. 4.3.4) which reduces the amount of light collected by the objective lens when observed in OM. A possible absorption of light in the photonic structure, which is not considered in the simulation, is maybe another reason for this difference in reflectivity. Also due to the strong scattering, the magnification dependence of the reflectivity can be explained by the fact that more light is collected, i.e. higher reflectivity, when the collecting angle is enlarged at higher magnification using an objective with larger numerical aperture. Most results of the optical experiments performed on the cuticle of the beetles (Fig. 4.3.1-3) show the characteristics of a flat multilayer reflector. However, none of the multilayer structure models would lead to the observed scattering even sometimes deviates from the specular reflection (Fig. 4.3.4b, d). The strong scattering may originate from the structural inhomogeneity in the multilayer structure or in the outer epicuticular layer located above the multilayer structure, such as the observed flakes and particles (Fig. 4.2.11, 12). It can also originate from the inherent curvature of the whole cuticle. To study which structural parameters will influence the reflectivity of the investigated photonic structure and how they do this, simulations using a model with unchanged constituting materials (i.e. unchanged refractive indices) but different structural parameters were carried out. The simulation results show that the spectra of a multilayer structure with varying thicknesses of the two sub-layers throughout the multilayer structure do not significantly differ from the spectra obtained for constant thicknesses, if the variation of the thickness is within the range of the measured standard deviation (Fig. 4.4.4). This indicates that the reflectivity spectra calculated based on the idealized model with constant layer thickness for each type of sub- layers can reproduce the characteristics of the real structure. The number of the bi-layers in the model has a significant influence on the maximum reflectivity of the photonic structures of the two beetles. The more bi-layers form the multilayer structure, the higher is the reflectivity (Fig. 4.4.3). Next, the influence of the thickness of the outermost layer (outer epicuticle) on the reflectivity and the width of the reflection peaks of the cuticle were studied. Simulations using different thicknesses of this layer show that both beetles obtain the

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Chapter 4 | Multilayer Structures (Ground Beetles) broadest peaks and lowest reflectivity when the thickness of the outer epicuticle is equal to the one observed in the beetles (Fig. 4.4.5b and Fig. 4.4.6b). Up to now, this effect has not been widely considered by the community studying structural coloration in nature, because most models of multilayer reflectors reported previously do not distinguish the thickness of the outermost layer from the similar sub-layers in the underlying multilayer structures. The study by Yoshioka et al. (2012) is the only one to our knowledge that has shown and considered the influence of the thicknesses of the outermost layers to the resulting optical properties of the multilayer structure of C. fulgidissima. However, in that case, the thicknesses of the outermost layers are modified to increase the reflectivity of the structure. In contrast, the effect of the outermost layers of the two investigated ground beetles here is to decrease the reflectivity. It is known for the synthetic multilayer reflectors that the thickness of the outermost layer has a significantly impact on the reflectivity of the multilayer structure when the number of the layers in the stack is not infinite (Baumeister, 1999; Carniglia, 2000), which is the case for the two investigated beetles species. From a biological point of view, it makes sense to reduce the reflectivity and thus minimize the danger to be observed by potential predators. The model is further complicated by introducing the transparent wax layer on top of the outermost layer. In general, the reflectivity of the structure increases by adding the wax layer independently from its thickness (Fig. 4.4.7). However, even the highest reflectivity (black curve in Fig. 4.4.7) achieved by adding the wax layer to the model with the outermost layer being 101 nm thick (which corresponds to its real thickness in C. auronitens) is still lower than the reflectivity of a model with generic thicknesses of the outermost layer of e.g. 21 nm or 191 nm but without this wax layer (Fig. 4.4.5b). This result shows that the thickness of the outermost layer is a crucial parameter to the resulting reflectivity of the entire photonic structure, even if an additional wax layer is laid on top of it. Nevertheless, although the wax layer inevitably increases the reflectivity of the cuticle, this layer is necessary for the beetle to prevent itself from desiccation. Therefore the epicuticular structure of the two investigated beetles revealed in this study, including the outer epicuticle, multilayer structure, the flakes and particles and the wax layer, represents the evolutionary result of the necessity to fulfill the

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Summary requirements of two different biological functions: optical appearance and the permeability of the cuticle.

4.6. Summary

The primary origin of the greenish color reflected by the cuticle of Carabus auronitens and C. auratus is the multilayer structure forming their inner epicuticle. This structure consists of periodically alternating thin and thick layers of which the constituting materials are most probably melanin associated with proteins and sclerotized structural lipids, respectively. These different constituents result in different refractive indices of these two layer types. Although the specific refractive index for each layer type is difficult to determine, the average refractive indices of the photonic structures of C. auronitens and C. auratus were optically determined to be 1.70 and 1.77, respectively. The larger index of C. auratus is the main reason that it reflects light with longer wavelengths than C. auronitens does. Simulations based on the multilayer structure models of these two beetle species show that their reflectivity is largely influenced by the thickness of an outermost layer situated on top of the multilayers and also by the thickness of the wax layer external to this layer. The outermost layers formed by both beetles possess the optimal thicknesses to obtain the lowest reflectivity concurrent with the broadest reflection peaks for the cuticle. The additional wax layer inevitably increases the reflectivity of the cuticle, but this layer is essential for the beetles to prevent themselves from desiccation. This finding implies that the most advantageous reflectivity and color saturation for these two beetle species is adjusted by controlling the thickness of the layer situated under the wax layer. Although it is known that the thickness of the outermost layer has a significantly impact on the reflectivity of synthetic multilayer structures, this is one of the few studies that this effect is found and described in the multilayer reflectors of beetles. The experimental measurements show that the actual reflectivity of the beetle cuticle is lower compared to the simulation results and

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Chapter 4 | Multilayer Structures (Ground Beetles) strong diffusive scattering occurs. Both phenomena can be attributed to the scattering from the observed structural inhomogeneities, such as the lens-shaped particles or flakes on top of the multilayer reflectors of these two beetle species. The unique, well oriented and densely packed flakes found in C. auratus can also act as an additional permeability barrier which most probably represents an adaptation to the more arid habitats of this thermophilic species. The absorptive melanin molecules presumably present in the multilayer structures would represent another reason for the low reflectivity of the cuticle.

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

Chapter 5. Helicoidal Photonic Structures (Scarab Beetles)

5.1. Introduction

In addition to the ability to selectively reflect light of different wavelengths, the cuticle of Scarab beetles (Scarabaeidae) is able to reflect circularly polarized light (Michelson, 1911; Neville and Caveney, 1969; Caveney, 1971; Goldstein, 2006). The mechanism behind this optical phenomenon is that the helicoidal structure of the exocuticle of Scarab beetles acts as an optical analogue of cholesteric liquid crystals. Most of the examined species of Scarab beetles selectively reflect left handed circularly polarized light (Neville and Caveney, 1969), with exceptions of rare cases that the cuticle of some species like Plusiotis resplendens can reflect both left and right circularly polarized light at different spectral regions (Caveney, 1971). There are also Scarab beetles (e.g. Heterorrhina species) that do not selectively reflect circularly polarized light of any handedness (Neville and Caveney, 1969). Neville and Caveney (1969) showed that the exocuticles of these species do not have the typical helicoidal structure of insect cuticles. Instead, the structure of these exocuticles make them behave like multilayer interference reflectors consisting of alternating chitin/protein plates and another type of plates with a presumably different refractive index. However, the chemical composition of this second type of plates is unknown. In addition, these exocuticles contain a system of rods with a diameter of about 500 nm formed by chitin/protein fibers pervading the exocuticle perpendicularly with respect to the surface of the cuticle (Neville and Caveney, 1969; Neville, 1975). Recently, Scarab beetles with similar cuticular structures have been studied due to their unusual optical properties (Biró et al., 2010; Liu et al., 2011). Biró et al. (2010) have shown that the epicuticle (most likely, this is a misinterpretation and should be exocuticle like in other Scarab beetles) of the beetle Trigonophorus rothschildi varians contains a multilayer structure consisting of periodically stacked chitin layers with air gaps between

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Introduction two adjacent layers. This multilayer structure is interrupted by randomly distributed chitin rods which run through 10 to 20 periods of the layers. The cuticle of this species displays colors consisting of an unsaturated specular and a saturated non-specular component of the reflected light, which is unexpected for a standard multilayer reflector. The unsaturated white reflectance at normal incidence is attributed to the topmost, unstructured glassy wax layer of the epicuticle. Liu et al. (2011) have shown that the cuticle of the beetle Heterorrhina sexmaculata also contains a similar multilayer structure pervaded by rod-shaped structures, but the two types of constituting layers were assumed to be formed by chitin and melanoprotein, respectively. The material forming the rods was assumed to be melanoprotein as well. Similar to the unsaturated specular reflectance of the cuticle of T. rothschildi varians (Biró et al., 2010), the specular reflectivity of the cuticle of H. sexmaculata at normal incidence is almost uniform over the whole visible frequency range (Liu et al., 2011). In this case, this phenomenon is attributed to the specular reflection for all visible light wavelengths from layers with thicknesses systematically changing from 50 nm to 500 nm (Liu et al., 2011), which are located under the aforementioned multilayer structure. The wide angular dispersion of diffraction of the cuticle of H. sexmaculata, which is also observed in T. rothschildi varians (Biró et al., 2010), is induced by the disorder of the spacing between rods (Liu et al., 2011). Comparing these three studies on the Scarab beetles which do not selectively reflect circularly polarized light, it becomes obvious that the description of the cuticle structure, the assignment of chemical composition of the constituting materials and the interpretation of the optical properties are not consistent. In order to shed some light on these problems, I therefore compare the photonic structures and the optical properties of the cuticles of two Scarab beetle species: one is Cetonia aurata (Fig. 5.1.1a) whose cuticle selectively reflects left circularly polarized light (Neville and Caveney, 1969) and the other one is Dicranorhina oberthuri (Fig. 5.1.1d) whose cuticle does not reflect circularly polarized light. The lack of the polarization ability for the cuticle of D. oberthuri is revealed by its almost zero reflection when observed between crossed polarizers under an optical microscope (OM) (Fig. 5.1.1f).

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

Figure 5.1.1: Morphology of the two Scarab beetle species investigated in this study: C. aurata (a) and D. oberthuri (d). Optical micrographs of the elytra of C. aurata (b) and D. oberthuri (e) taken in bright field mode showing the orange and green coloration, respectively. Micrographs of the same region in (b) and (e) taken between two crossed polarizers under OM showing dark red coloration (c) and almost no light reflected (f), respectively. The white arrows in (c, f) indicate that the polarizer (P) and analyzer (A) are cross-polarized.

5.2. Structural characterization

5.2.1. Cross- and transverse-sectional surfaces of fractured samples Fracture surfaces exposing the cross-section of the cuticle of C. aurata show densely stacked thin layers in the exocuticle and thicker layers formed by bundles of fibers in the endocuticle (Fig. 5.2.1a). The total thickness of the exocuticle is about 15 µm. The stacking heights of the layers in the lower region of the exocuticle are not constant. The thicknesses of these layers, which can be up to about 1 µm, are larger than those of the layers in the upper region of the exocuticle (Fig. 5.2.1a). The cuticle of D. oberthuri has a similar general structure like

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Structural characterization the one of C. aurata, namely thin layered structures in the exocuticle and thick layers in the endocuticle (Fig. 5.2.1b). The thicknesses of the layers in the lower exocuticle, which can be up to 1.3 µm, are also larger than those of the layers in the upper region. The total thickness of the exocuticle of D. oberthuri is about 28 µm (Fig. 5.2.1b), which is almost twice as thick as the one of C. aurata. The most noticeable structural feature of the exocuticle of D. oberthuri, which makes it different from the one of C. aurata, is that the exocuticle is pervaded by distinct rod-shaped structures oriented perpendicular to the surface (Fig. 5.2.1b).

Figure 5.2.1: Scanning electron microscopy (SEM) images showing the fractured cross-sectional surfaces of the cuticle of C. aurata (a) and D. oberthuri (b). (exc: exocuticle; enc: endocuticle). High resolution SEM images show that these rods are filled with solid biological material (Fig. 5.2.2b). The thickness of these rods is 472 ± 35 nm and remains constant through the depth of many layers in the exocuticle (Fig. 5.2.2b). At the boundary between the lower region of the exocuticle and the upper region of the endocuticle, these rods fuse into the large pore canals present in the thick chitin/protein fiber - 76 -

Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles) planes (Fig. 5.2.1b and Fig. 5.2.2d). Another structural feature that makes the exocuticle of D. oberthuri different from the one of C. aurata is that the upper region of the exocuticle of D. oberthuri is formed by two types of layers displaying different appearances after fracture (Fig. 5.2.2c). One type of layer has a rigid, fibrous appearance whereas the other type is smooth and appears always less prominent with respect to the fracture plane. The thicknesses of both types of the layers are about 75 nm. The material of the smooth layers curves inwards, which makes them appear to be porous (Fig. 5.2.2c). As a result, the upper exocuticle of D. oberthuri (Fig. 5.2.2b) appears to be less densely stacked than that of C. aurata (Fig. 5.2.2a). The layers in the lower region of the exocuticle of D. oberthuri have a typical twisted plywood structure, which is different from the structure of the layers in the upper region (Fig. 5.2.2d). From the fracture surface, it is difficult to distinguish the epicuticle of C. aurata from its exocuticle (Fig. 5.2.2a). The epicuticle of D. oberthuri differs from its exocuticle in the absence of the alternating two types of layers and the rods system (Fig. 5.2.2b). The transverse-sectional fracture surface of the upper exocuticle of C. aurata shows fibrous structures forming arc-shaped features (Fig. 5.2.3a). At the deeper level of the upper exocuticle, the fibers are coarser and holes with diameters of about 90 nm are observed (indicated by white arrows in Fig. 5.2.3b). The fractured surface of the upper exocuticle of D. oberthuri in the same view (Fig. 5.2.3c) revealed round shapes distributed in the cuticle with diameters of about 490 nm and a center to center distance of 1 ± 0.16 µm (Fig. 5.2.3c). From the dimensions and distribution of these round shaped features, they correspond to the top view of the fractured rods which are oriented perpendicular to the surface. These rods are surrounded by planar fibrous structures with curved trajectories (Fig. 5.2.3d). The smooth layers observed in the cross-sectional fracture surface of cuticle (Fig. 5.2.2c) appear as dark contrasted, recessed areas in between two of the curved planes in this top view of the exocuticle (Fig. 5.2.3c, d).

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Structural characterization

Figure 5.2.2: High resolution SEM images showing the fractured cross- sectional surfaces of the cuticle of C. aurata (a) and D. oberthuri (b-d). (a) and (b) showing the epicuticle and upper exocuticle region of C. aurata and D. oberthuri, respectively. (c) Micrograph showing two types of layers (fibrous layer and smooth layer) in the upper exocuticle region of D. oberthuri. (d) The pore canals in the lower exocuticle region of D. oberthuri fuse into one larger pore canal in the endocuticle.

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

Figure 5.2.3: SEM images of horizontally fractured surfaces of the exocuticle of C. aurata (a, b) and D. oberthuri (c, d). (b) Micrograph showing a fractured surface at a deeper level of the exocuticle of C. aurata than (a). The white arrows in (b) indicate the locations of pore canals.

5.2.2. Section surfaces of microtomed samples The ultramicrotome polished cross-sectional surface of the cuticle of C. aurata shows that the exocuticle is about 13 µm thick and formed by 58 horizontally stacked layers (Fig. 5.2.4a). The exocuticle can be subdivided into four regions (from top to bottom, regions I – IV in Fig. 5.2.4a) according to the variations of the thicknesses of the constituting layers in each region. The region I is about 5 µm in thickness and consists of 28 layers with a constant thickness of 181 ± 8 nm (Fig. 5.2.4a and Fig. 5.2.5a). The adjacent underlying region II is about 3.1 µm in thickness, which is formed by 13 layers with gradually increasing layer thickness from about 200 nm to 300 nm (Fig. 5.2.4a). In the next region III, the horizontal layers are perforated by vertical structures with a thickness of about 500 nm (Fig. 5.2.4a).

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Structural characterization

This region is about 2 µm in thickness and consists of 10 layers whose thicknesses are varying from 150 nm to 250 nm (Fig. 5.2.4a). The lowest region IV of the exocuticle is about 3.3 µm in thickness formed by 7 layers with much larger layer thicknesses than those of the above regions, gradually increasing from 0.30 µm to 1.00 µm (Fig. 5.2.4a). The whole exocuticle of C. aurata is covered by an epicuticular layer of 424 ± 16 nm in thickness (Fig. 5.2.4a and Fig. 5.2.5a). The polished vertical cross-sectional surface of the cuticle of D. oberthuri shows that its exocuticle is about 28 µm thick (Fig. 5.2.4b) which is more than twice thicker than the one of C. aurata (Fig. 5.2.4a). The number of the constituting layers in the exocuticle of D. oberthuri is 123 (Fig. 5.2.4b), which is also two times more than the one of C. aurata (Fig. 5.2.4a). Another major difference of the general structure between the exocuticle of the two beetles is that the vertical rod-shaped structures are visible through the whole exocuticle for D. oberthuri (Fig. 5.2.4b) whereas it is only visible in the lower part of the exocuticle of C. aurata (region III, Fig. 5.2.4a). Similar to C. aurata, the exocuticle of D. oberthuri can also be subdivided into three regions (from top to bottom, regions I – III in Fig. 5.2.4b). The region I is the thickest one among the three regions (about 18 µm thick) and consists of 97 layers with a constant layer thickness of 171 ± 11 nm (Fig. 5.2.4b). The underlying region II is about 5 µm in thickness and is formed by 20 layers with layer thicknesses varying from 190 nm to 335 nm which are larger than those of the layers in region I (Fig. 5.2.4b). The lowest region III is about 4.5 µm thick and consists of 6 layers with gradually increasing thickness from 0.37 µm to 1.30 µm (Fig. 5.2.4b). The exocuticle of D. oberthuri is covered by an epicuticular layer of 920 ±39 nm in thickness (Fig. 5.2.4b and Fig. 5.2.5b), which is two times thicker than the one of C. aurata (Fig. 5.2.4a and Fig. 5.2.5a). The epicuticle of D. oberthuri appears to consist of two layers (Fig. 5.2.5b).

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

Figure 5.2.4: SEM images of ultramicrotome polished cross-sectional surfaces of the cuticle of C. aurata (a) and D. oberthuri (b). The exocuticle (exc) of C. aurata is subdivided into four regions (I-IV) and three regions (I- III) for D. oberthuri according to the variations of the thicknesses of the constituting layers. (epc: epicuticle; enc: endocuticle).

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Structural characterization

The cross-sectional surfaces of the region I in the exocuticle of D. oberthuri show pores at the boundaries between the vertical rod- shaped structures and the horizontal layers, which can be regarded as an artifact from the mechanical polishing (Fig. 5.2.4b and Fig. 5.2.5b, c). The layers in the region I are often not perpendicular to the rods and thus not parallel to the cuticle surface (Fig. 5.2.5c, the vertically aligned pores indicate the position of the rods). The layers in the vicinity of the rods (within a radius of about 500 nm) are often not at the same level of the layers in between two rods, thus forming an interlocked finger-like structure (Fig. 5.2.5d). The cuticle of D. oberthuri was then polished at an oblique angle of about 45° with respect to the surface and was observed perpendicular to this surface (Fig. 5.2.6). From this view, the upper region of the exocuticle of D. oberthuri shows a layered structure interrupted by oval shaped features which are the resulting oblique sections of the rods after polishing (Fig. 5.2.6). The layers at the upper and lower margins of the oval shaped features appear inclined with respect to the layers in between the ovals at an angle of about 15° (Fig. 5.2.6).

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

Figure 5.2.5: High resolution SEM images showing ultramicrotome polished cross-sectional surfaces of the cuticle of C. aurata (a) and D. oberthuri (b-d). (a) and (b) showing the epicuticle and upper exocuticle region of C. aurata and D. oberthuri, respectively. (c, d) showing the rod- shaped structures and the interlocked finger-like structure formed by the apparently oblique lamellae of D. oberthuri.

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Structural characterization

Figure 5.2.6: SEM image showing the exocuticle of D. oberthuri polished at an oblique angle of about 45° with respect to the surface viewed perpendicular to this polished surface. The cuticles of both beetles were also ultramicrotome polished horizontally almost parallel to the cuticle surface (see Fig. 5.2.7, in a similar angle of the view of the cuticle as shown in Fig. 5.2.3). The polished surface of the upper exocuticle of C. aurata shows numerous narrow pores (cross sections of pore canals) with a thickness of about 15 nm forming arc-shaped patterns which repeat in a periodic manner (Fig. 5.2.7a). The arc-shaped patterns also appear on the polished surfaces of the upper exocuticle of D. oberthuri (Fig. 5.2.7b, c). However, here the structural elements responsible for the appearance of these patterns are the sections of the rods. Because of the much larger dimensions of the sections of the rods compared to those of the pores observed in C. aurata, the number of the rods necessary to generate an artificial arc is much smaller in D. oberthuri. The sufficient number can be around three (Fig. 5.2.7b) or even only one (Fig. 5.2.7c) for individual arcs.

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

Figure 5.2.7: SEM images of the oblique sections of the upper exocuticle of C. aurata (a) and D. oberthuri (b, c) showing arc-shaped patterns formed by the cross section of the pore canals of C. aurata (a) or the rod like structures of D. oberthuri (b, c). Optical micrographs in bright field mode of the horizontally polished cuticle surfaces of both beetles show vivid green to orange colored layers near the surface of the cuticle. These layers are located above the more brownish deeper layers (Fig. 5.2.8a, c). When the cuticle of C. aurata is observed with a linearly polarized filter, the colored layer shows clear undulating striations (Fig. 5.2.8b, about 40 layers) which are not observed in bright field mode (Fig. 5.2.8a). No such effect is observed for the colored layers of D. oberthuri with the same filter setup (Fig. 5.2.8d). For D. oberthuri, the arc-shaped patterns aforementioned are often visible (Fig. 5.2.8c, d).

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Structural characterization

Figure 5.2.8: Optical micrographs of oblique sections of the cuticle of C. aurata (a, b) and D. oberthuri (c, d) taken without (a, c) or with (b, d) a linear polarizer.

5.2.3. Structure changes of NaOH treated samples In order to study possible differences in the chemical composition of the two types of layers present in the upper exocuticle (region I in Fig. 5.2.4b) of D. oberthuri, I treated the polished cuticle with NaOH solution (1.5 M) for 1 day. Compared to the relatively smooth cross- sectional surface of the cuticle before treatment (Fig. 5.2.5b, d), the treated cuticle shows pores between layers indicating that one type of layer is washed out more than the other one (Fig. 5.2.9a, b). The thickness of the remaining layers varies from 80 nm to 150 nm. The majority of the material forming the rods remained. The layers in the

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles) vicinity of the rods often discontinue from the layers in between the rods (Fig. 5.2.9a, b). The epicuticular layer of D. oberthuri is still present after the treatment, but its thickness decreases to 648 ± 26 nm (Fig. 5.2.9a). The subdivision of the epicuticle into two layers is clearly visible after this treatment (Fig. 5.2.9a).

Figure 5.2.9: SEM images showing ultramicrotome polished cross-sectional surfaces of the cuticle D. oberthuri after exposure to NaOH solution (1.5 M) for 1 day.

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Optical characterization

5.3. Optical characterization

5.3.1. Reflectance at normal incidence The reflectivity spectra of the elytra of both beetles at normal incidence were measured under OM using objectives with different magnifications. In general, the positions of the peaks of both beetles gradually shift to shorter wavelengths and the reflectivity of the peaks increases up to 0.27 for C. aurata and 0.18 for D. oberthuri as the magnification of objectives increases (Fig. 5.3.1a, b). For the magnifications of 50 × and 100 ×, the maximum reflectivity peaks of C. aurata locate at 593 nm and 586 nm (Fig. 5.3.1a), whereas those of D. oberthuri locate at shorter wavelengths of 553 nm and 547 nm, respectively (Fig. 5.3.1b). For C. aurata, one is already able to recognize one peak at 599 nm with a maximum reflectivity of 0.016 at the magnification of 5 × and a well-defined peak at 597 nm with a higher maximum reflectivity of 0.16 at 20 × (Fig. 5.3.1a). For D. oberthuri, in contrast, there are no peaks well recognizable at magnifications from 5 × to 20 × (Fig. 5.3.1b).

Figure 5.3.1: Reflectivity spectra of the elytra of C. aurata (a) and D. oberthuri (b) at normal incidence measured under OM using objectives of different magnifications indicated by differently colored curves.

5.3.2. Polarization effect In order to study the different polarization effects of the cuticle of these two beetles, the reflection spectra were measured under the condition that the polarization state of the linear polarizer for the incident light is perpendicular to the one of the analyzer for the

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles) reflected light (cross-polarization, indicated by sp or ps in Fig. 5.3.2) at different angles of incidence (50° and 70°). The recorded spectra show well defined high intensity peaks for C. aurata at 535 nm for 50° and 493 nm for 70° (Fig. 5.3.2a), but no peaks for D. oberthuri for both angles (Fig. 5.3.2b).

Figure 5.3.2: Wavelength-dependent intensity of reflected light of C. aurata (a) and D. oberthuri (b) measured at different combinations of the polarization states of the polarizer and analyzer. (s: s polarization; p: p polarization; sp or ps: cross-polarization) at different angles of incidence (50° and 70°).

5.4. Discussion

5.4.1. Photonic structures of the Scarab beetle species The optically active exocuticle of C. aurata (region I in Fig. 5.2.4a) is formed by a helicoidal structure as reported previously (Neville and Caveney, 1969), which is confirmed by the arc-shaped patterns observed in both horizontally fractured (Fig. 5.2.3a) and polished (Fig. 5.2.7a) surfaces. The diameter of the pore canals in the upper region of C. aurata’s exocuticle is about 15 nm (Fig. 5.2.7a) and increases to about 90 nm in lower regions (Fig. 5.2.3b). In the region further below (region III in Fig. 5.2.4a), these small pore canals fuse to form larger pore canals with a width of about 500 nm. The major difference between the exocuticle of D. oberthuri and C. aurata is the presence of rod-shaped structures pervading the exocuticle of D. oberthuri perpendicularly to the surface (Fig. 5.2.1b and Fig. 5.2.4b). The

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Discussion widths of these rods (around 500 nm) do not vary as much as and are much larger than those of the pore canals in the exocuticle of C. aurata. Neville (1975) termed these rods as Cuticular Rods distinguishing from Pore Canals where he introduced the vertically oriented structures occurring in the arthropod cuticle. My observations show that these rods directly fuse into the larger pore canals in the endocuticle (Fig. 5.2.1b and Fig. 5.2.2d). Therefore, these rods are most probably pore canals filled with solid biological materials which are believed to be chitin/protein complexes (Neville, 1975; Biró et al., 2010) or melanoprotein (Liu et al., 2011). The chemical treatment with NaOH solution revealed that the majority of the materials in the rods did not dissolve (Fig. 5.2.9). This result implies that the material forming the rods is not likely to be melanoprotein, which is known to dissolve during alkali treatment (Schultz and Rankin, 1985b). It is then more likely that these rods are formed by chitin/protein fibers oriented perpendicularly to the surface, due to the fibrous appearances of the materials within the rods (Fig. 5.2.2b and Fig. 5.2.5b, d). These rods have a round horizontal cross-section (Fig. 5.2.3c, d and Fig. 5.2.7b, c) and a constant width throughout many layers in the exocuticle (Fig. 5.2.2b), which indicates that three dimensionally these rods have a cylindrical shape. This shape is different from the typical twisted ribbon shape of pore canals (Neville and Berg, 1971, see also Section 2.2; Neville, 1975). The horizontal cross-section of a classical pore canal shows an elliptical shape of which the long axis follows the orientation of the surrounding nanofibrils (Neville, 1975). However, here, the nanofibrils are curved round the pore canals (Fig. 5.2.3c, d) instead of forcing the shape of the pore canals to follow the orientations of the nanofibrils, probably due to the solid materials inside the pore canals in the exocuticle of D. oberthuri. The helicoidal rotation of the curved instead of straight nanofibrils in the horizontal chitin/protein fiber layers results in apparent obliquely stacked layers with respect to the cuticle surface (Bouligand, 1972), which is most probably the reason of the obliqueness of the layers observed in the vicinity of the rods of D. oberthuri (Fig. 5.2.5c, d and Fig. 5.2.6). Another major difference between the exocuticle of D. oberthuri and C. aurata is that the former one showed two types of layers with similar layer thicknesses but different appearance and alternating shapes, protruding or recessed, upon fracturing (Fig. 5.2.2b, c). These

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles) two types of fracture surfaces can be explained if one assumes the materials in each type of layers are different and hence also the behavior upon fracturing and drying of these two types of layers. This assumption is supported by previous transmission electron microscopy (TEM) analysis of the exocuticle of other closely related Scarab species where two types of layers with different electron contrast were observed and were suggested to consist of different materials as well (Neville and Caveney, 1969). In contrast to the undulating fracture surfaces, the polished surfaces of the exocuticle of D. oberthuri appear to be smooth and resemble the polished surfaces of C. aurata (compare Fig. 5.2.5a and b), which gives an impression that chitin/protein layers are the only layers forming the exocuticle of D. oberthuri. However, this smooth surface is probably an artifact of polishing which mechanically smeared the edges of the original fibrous layers and forced the materials into the depressions originating from the smooth layers. NaOH treatment of the polished exocuticle surfaces of D. oberthuri revealed that the material of one type of the two layers is relatively easier to be dissolved than the other one, thus those appear as pores (Fig. 5.2.9). This can be explained by the difference in the chemical composition of the materials in these two types of layers. However, it cannot be excluded that it is due to material smeared by polishing which is not strongly bonded to the depressions originating from the smooth layers and thus prone to be washed away. Nevertheless, both explanations imply that the exocuticle of D. oberthuri consists of a second type of layer formed by materials different from that of chitin/protein layers. Due to their small dimensions, the exact chemical composition of this second type of layer is difficult to be determined, and hence is still unknown at the current state. However, this second layer is not likely to be an air gap between the chitin/protein layers as suggested by Biró et al. (2010), since no immediate color change was observed when a broken cuticle of D. oberthuri was immersed into water that would fill the air gaps and change the effective refractive index of the cuticle. In addition to this hypothesis of two types of layers in the exocuticle of D. oberthuri, the occurrence of helicoidal structures in the exocuticle as well was suggested by the observations of arc-shaped patterns from both the SEM (Fig. 5.2.7b,c) and OM (Fig. 5.2.8c,d) images of the polished samples. How the aforementioned second layer type is formed

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Discussion concurrently with the formation of helicoidal chitin/protein layers and the rod-shaped structures is an interesting topic for further investigation, and of high importance for the understanding of the unusual optical properties of the exocuticle of D. oberthuri and other similar Scarab species.

5.4.2. Optical properties of the photonic structures The exoskeleton of D. oberthuri does not selectively reflect circularly polarized light like C. aurata does, since no reflection was observed (Fig. 5.1.1f) and measured (Fig. 5.3.2b) under cross-polarization. This is most probably due to the different microstructure of the exocuticles of D. oberthuri compared to the classical helicoidal structure of the cuticle of C. aurata. Since the photonic structure of C. aurata is based on the helicoidal structure, the average refractive index of this structure can be estimated by the equation = / = 1.65 (p = 2a, see Section 2.3.3), where λ (597 nm) is the wavelength of maximum reflectivity peak at normal incidence (Fig. 5.3.푛� 1a),휆 p푝 is the thickness of one pitch and a (181 ± 8 nm) is the stacking height of the helicoidal structure measured from the vertically polished surface of the exocuticle (Fig. 5.2.5a). This average refractive index is very close to the value (1.66) estimated from the refractive indices measured in the cuticle of other Scarab beetles (Plusiotis, see Section 2.2) (Caveney, 1971). The photonic structure of D. oberthuri is more complicated by the presence of the vertical rod structures and a second type of layer. However, the rod structures should not have an influence on the color of the exoskeleton of D. oberthuri, since the diameter (472 ± 35 nm) and the center to center distance (1 ± 0.16 µm) of these rods are too large to affect the wavelengths of the reflected visible light. Therefore, for estimation of the average refractive index of the photonic structure of D. oberthuri, this structure can be approximated as a multilayer structure. Then the average refractive index can be calculated by = /2 = 1.62, which is smaller than the one of C. aurata, where λ = 553 nm (Fig. 5.3.1b) and a = 171 ± 11 nm (Fig. 5.2.5b). This decrease푛� 휆 푎of the average refractive index can be explained by the occurrence of the second type of layer in the D. oberthuri compared with C. aurata. The refractive index of this second type of layer is then most probably smaller than the one of chitin/protein layers.

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

The specular reflectivity of the elytra of D. oberthuri at approximately normal incidence (small numerical apertures) is almost uniform over the whole visible frequency range, but shows saturated reflections when objectives with larger numerical aperture were used to measure the spectra and thus under partially oblique incidence (Fig. 5.3.1b). This optical phenomenon is unusual for both multilayer structures and helicoidal structures and was previously reported for two other Scarab species with similar rod structures (Biró et al., 2010; Liu et al., 2011). For T. rothschildi varians, this phenomenon was attributed to the waxy layer on top of its multilayer structure (Biró et al., 2010). However, our simulation (Fig. 4.4.6 in Section 4.4.6) showed that waxy layers with different thicknesses only have an influence on the reflectivity of the multilayer structure, but do not change the hue of the reflected light. Therefore, it is not likely that the unsaturated specular reflection of D. oberthuri is due to its waxy layer. For H. sexmaculata, this phenomenon is attributed to the specular reflection, which covers all visible light wavelengths, originating from the layers with thicknesses changing from 50 nm to 500 nm (Liu et al., 2011). Here, for D. oberthuri, the thicknesses of all layers in different regions of the exocuticle are equal to or larger than 171 nm, which theoretically should lead to the estimated maximum reflectance of all layers above 553 nm. Therefore, the measured uniform reflections (Fig. 5.3.1b) should not be the result of the overlap of reflections from layers with different thicknesses in the exocuticle of D. oberthuri. In addition, this concept cannot explain the reemergence of the saturated reflections measured under oblique incidence. To fully understand the optical properties of the exocuticle of D. oberthuri and how they relate with the complex photonic structure, a theoretical study is needed based on a deeper understanding of its structure.

5.5. Summary

Different structural arrangement of the chitinous fibrils forming the exocuticles of Cetonia aurata and Dicranorhina oberthuri are responsible for the coloration of these two beetles. C. aurata forms very regular helicoidal cuticular structure that can selectively reflect circularly polarized light. In contrast, the photonically active

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Summary exocuticle of D. oberthuri is formed by horizontally layered structures pervaded by an additional vertical rod system. These rods are pore canals filled with chitin/protein fibers. The pore canals have a cylindrical shape instead of the typical twisted ribbon shape. Due to their relatively large dimensions (about 500 nm in diameter), these rods are not expected to affect the color of the reflected visible light. For the horizontally layered structure, arc patterns observed on oblique sectional surfaces indicate the existence of helicoidal arrangement. Besides the fibrous chitinous layers, another type of layer coexists in the photonic structure of D. oberthuri, which shows different material properties upon fracturing and dissolving by NaOH solution. The chemical composition of this second type of layer is unknown, but its refractive index is most probably smaller than the one of the chitin/protein complex. The correlation between these two types of alternatively stacked layers and their relation with the rod system is still not fully understood. In contrast to the helicoidal arrangement in C. aurata, this complicated structure is not analogous to a cholesteric liquid crystal and thus cannot selectively reflect circularly polarized light. One unusual optical property of the cuticle of D. oberthuri is that its specular reflectivity is almost uniform over the whole visible frequency range at approximately normal incidence. Hence, it appears to be silver bright. However, the measured spectra become saturated in the green region under oblique incidence. This unique optical effect neither occurs in multilayer structures nor in helicoidal structures. It is safe to assume that it is related to the scattering of the additional rod system. It is not likely to originate from scattering of the wax layer or the broad reflection of light by the layers with systematically varying thicknesses as suggested in literature on Scarab beetles with similar structures. The complicated photonic structure in the exocuticle of D. oberthuri is still not fully understood at the current state. Here, additional characterization methods like TEM tomography are required to provide three-dimensional information of the structure. Recently, helicoidal photonic structures as occurring in the exocuticle of Scarab beetles have been biomimetically fabricated using self- organizing and self-aligning liquid crystal polymers (Matranga et al.,

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Chapter 5 | Helicoidal Photonic Structures (Scarab Beetles)

2013). It would be interesting to investigate whether a system of vertical rods like those in the exocuticle of D. oberthuri can be incorporated into the synthetic material and whether the optical properties would be influenced in an analogous fashion. In addition, this biomimetic approach would us help to understand the impact of the rod system on the optical properties of the photonic structure of this beetle species. Whether and how the rod system in the exocuticle of D. oberthuri influences the mechanical properties of the cuticle is also an interesting topic, since the exocuticle is a mechanically relevant part of the exoskeleton.

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

Chapter 6. Three-dimensional Photonic Crystals (Weevils)

6.1. Introduction

Many species in the group of weevils or snout-beetles (Curculionidae) produce their often vivid colors with various three-dimensional (3D) photonic structures that have developed inside scales located on the surface of their exoskeletons (Parker et al., 2003; Welch et al., 2007; Galusha et al., 2008; Pouya et al., 2011; Wilts et al., 2011; Wilts et al., 2012; Wu et al., 2013). Among these beetles, the weevil genus Entimus includes seven species whose integuments are covered with green, blue and golden iridescent scales, with the exception of E. arrogans which has only whitish scales (Morrone, 2002). These species are distributed in the Neotropical region, from Mesoamerica to northeastern Argentina (Morrone, 2002). I investigated the scales of E. imperialis, of which the specimen was collected from southern Brazil. The otherwise black exoskeleton of this species is covered with greenish colored spots on the dorsal side of its body (Fig. 6.1.1a). These spots are formed by clusters of colorful scales located in circular shaped depressions on the elytra (hard forewings) (Fig. 6.1.1b) and in characteristically shaped domains on the surface of other body parts. Individual scales have just one or several well defined domains showing different colorations (Fig. 6.1.1c). The displayed colors cover nearly the full range of the visible spectrum. Additionally, I frequently observed scales with transparent domains through which the color of the scale located underneath could be observed (white arrow in Fig. 6.1.1d). The structure and color producing mechanism of scales from Entimus species has already been studied almost 100 years ago (Mallock, 1911; Michelson, 1911; Onslow, 1923; Mason, 1926). However, due to the technical limitations of microscopy at that time, the latest description of the microstructure of the scales was an interior lamellar structure enclosed by the cuticle (Mason, 1926). Recently,

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Introduction

Deparis and Vigneron (2010) have modeled the ultrastructure found in the scales of E. imperialis as a face-centered cubic structure resulting from the ABC stacking of perforated and corrugated chitin sheets and calculated the photonic response of this structure using the concept of stratified medium. Wilts et al. (2011) identified the photonic structure of this species to be a diamond-based bicontinuous cubic structure (BCS) mainly by matching transmission electron microscopy (TEM) cross-sectional images of the interior structure of the scales with cross sections derived from level surface models with varying parameter t (See also Section 3.6).

Figure 6.1.1: The neotropic weevil Entimus imperialis. (a) Habitus of the investigated specimen. (b) Optical micrograph of scales located in a depression on the dorsal surface of the otherwise black elytra. (c) Individual scale with three differently colored domains. (d) A scale with a transparent domain (arrow) through which the dark orange color of a subjacent scale is visible. I studied the structure of the scales of E. imperialis mainly using scanning electron microscopy (SEM) on ultramicrotome polished samples and focused ion beam (FIB) prepared samples. The main focus was the correlation of the ultrastructure of the 3D photonic crystals with their optical response, both, experimentally and theoretically. In addition, an unusual polarization transfer effect was observed mostly in the green domains of scales and I investigated its relationship with the orientation of the photonic structure. For scales with transparent domains, additional efforts were made in terms of - 98 -

Chapter 6 | Three-dimensional Photonic Crystals (Weevils) chemical analysis of the constituting materials in this type of domain by using energy dispersive X-ray (EDX) and Fourier transform infrared (FTIR) spectroscopies to explain this optical phenomenon.

6.2. Structural characterization

6.2.1. Intact and broken scales Complete scales which were removed from the exoskeleton of E. imperialis have an elongate-oval shape and are about 100 µm long and 30-60 µm wide (Fig. 6.2.1a, c). The upper side of the scale surface is structured by parallel grooves with about 2 µm interval oriented along the longitudinal axis (Fig. 6.2.1a, b). The longitudinal strips confined by the large grooves are convexly domed and their surface is structured with a feather-like pattern of fine grooves with intervals of about 150 nm (Fig. 6.2.1b). On the underside, which usually faces towards the exoskeleton before removal, the scales have a smooth and unstructured surface (Fig. 6.2.1c). The scales are outgrowths of the cuticle and are connected to the exoskeleton by a short, cylindrical stem (Fig. 6.2.1c). Removal of the scales from the exoskeleton results in fracture surfaces of the stem showing a radial arrangement of rough, fibrous material with interspersed porosities (Fig. 6.2.1d). Due to the forces exerted during harvesting the scales by scraping with a needle, some scales were cross-sectional fractured exposing the periodically structured core (Fig. 6.2.1b). In other cases, the shell of scales was partially stripped off exposing the superficial layers of the core (Fig. 6.2.2a). Occasionally, spherical voids were observed within the fine, periodically structured core (Fig. 6.2.2a). The spherical holes with diameters ranging from 800 nm to 2 µm are natural defects within the core and occur mostly in regions close to the stem or the tip of the scales. High resolution SEM images of fracture surfaces of the cuticular network forming the core revealed no evidence of fibrillar structures (Fig. 6.2.2b, c).

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Structural characterization

Figure 6.2.1: SEM images of the upper side (a, b) and underside (c) of the scales of E. imperialis. (b) Micrograph showing the microstructure on the upper surface and inside the scale. (d) Micrograph showing the cross-section of the stem of the scale in (c).

Figure 6.2.2: (a) SEM image showing the exposed photonic crystals in the core of a scale. (b, c) High resolution SEM images showing the microstructure present in the different domains in (a).

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

6.2.2. Microtome polished scales In order to expose the photonic structure in the scales for characterization efficiently, I applied ultramicrotome polishing to pieces of elytron cuticle where scales are present (with additional fixation using super glue) and also polishing to scales that were first harvested and subsequently fixed in super glue. On the polished cross- sections of the elytron, near the external surface of the cuticle, it was occasionally possible to observe sections through the stems of scales (Fig. 6.2.3, obliquely cut). The dim greenish reflection in the upper part of the light micrograph (lower right inset in Fig. 6.2.3) indicates the main body of this scale which is out of focus. The cuticular layers distanced away from the stem proceed parallel to the external surface. In the epicuticle, there is only one layer (about 500 nm in thickness) distinguishable, whereas the exo- and endocuticle form layers with different thicknesses (varies from 90 to 530 nm and 1.8 to 3.6 µm, respectively) (Fig. 6.2.3). Close to the stem, the fiber layers of the exocuticle curve inwards and form a cylindrical invagination that protrudes into the endocuticle and broadens slightly at its proximal end. Here, the layers curve in the opposite direction inside the cavity of the cylindrical bulge, where they form the cylindrical stem of the scale. The stem proceeds towards distal and penetrates the exo-and epicuticle to form the bulk of the scale that is located above the supporting exoskeleton. Presumably, the shell of the scales is formed by the exocuticular layers. To what extent the epicuticle contributes to the formation of scales could not be fully clarified due to the lack of a perfect perpendicular cut through a stem area. The endocuticular layers adjacent to the invaginating exocuticle become thinner and follow the orientation of the exocuticle. The proximal, thickened part of the stem where the layers curve inwards forms an anchoring structure that is located 5 to 7 layers deep (from distal to proximal) within the endocuticle (Fig. 6.2.3).

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Structural characterization

Figure 6.2.3: SEM image of an ultramicrotome polished cross-section through a scale stem at its insertion site within the cuticle of the elytra. The dashed lines indicate the boundaries between the epicuticle (epc), exocuticle (exc) and endocuticle (enc). The black arrows indicate the trajectory of the exocuticle surrounding the stem. Inset: Optical micrograph of the same region (sg: super glue used to fix the scales for polishing). Cross sections of scales show that they are generally oriented parallel to the external surface of the elytron and reveal a structured core enclosed by a continuous shell (Fig. 6.2.4). The average thickness of the entire scale is 6.6 ± 0.4 µm. The average thickness of the core amounts to 3.7 ± 0.4 µm. The shell forming the upper side of the scale is 1.6 ± 0.1µm thick, whereas the underside is slightly thinner at an average of 1 ± 0.2 µm. While the polished surface of the shell appears smooth, the core consists of a 3D network of struts and air. Optical micrographs of the exposed core of a scale shows two differently colored domains (upper left inset in Fig. 6.2.4), yellow in the upper right and blue in the lower left region. On SEM images of the same sample it becomes obvious that the shape and position of the colored

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) domains coincide perfectly with a change in the aspect of the 3D network. The shell does not reflect vivid colors like the core, but displays a brownish color similar to the bulk cuticle (upper left inset in Fig. 6.2.4). At higher magnification, one can observe the seamless transition from one type of 3D network to another in the region near the boundary between the two domains (lower right inset in Fig. 6.2.4). This micrograph also revealed that, within the shell of both sides of the scale, layers with an average thickness of about 180 nm are presented adjacent to the core (lower right inset in Fig. 6.2.4). The number of layers observed was about 5 in the shell of upper side of the scale and only 2 in the undersdie. The thickness of the outermost layer forming the scale surface is about 1µm for the upper side and 600nm for the underside.

Figure 6.2.4: SEM images of ultramicrotome polished cross section through the scales. Upper inset: Optical micrograph of the same region showing the color appearance. Lower inset: high resolution SEM image showing the microstructure in a local region of one scale including the shell and the core.

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Structural characterization

The top surface of the core was exposed (bright contrasted region in the middle of Fig. 6.2.5b) by oblique cutting through the shell using ultramicrotome. No significant color change is observed in this exposed region when comparing the same scale before (Fig. 6.2.5a) and after (Fig. 6.2.5c) the removal of the shell under the light microscope. Under oblique cutting, the shell of the upper side of scales usually shows several inner layers with jagged edges and a homogeneous outer layer (Fig. 6.2.5d).

Figure 6.2.5: Optical micrographs of one scale before (a) and after (c) microtome polishing parallel to the surface. (b) SEM image showing the same scale as in (c) with exposed structured core. (d) High resolution SEM image showing the fibrous layered structure in the shell of the upper side of the scale.

6.2.3. Focused ion beam (FIB) milled scales In order to expose the photonic structures in individual domains of interest, I used FIB to mill off the shell in both perpendicular and

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) parallel directions to the surface of scales (Fig. 6.2.6b, see also Fig. 3.3.1). In scales where the photonic crystals were exposed (Fig. 6.2.6c), I observed distinct structural domains with different periodic patterns whose shapes and boundaries correspond exactly to differently colored domains observed with optical microscope (OM) (Fig. 6.2.6a). SEM images of the edges created by milling in two perpendicular directions show that the 3D periodic patterns are constituted by solid struts forming fourfold ramifications (Fig. 6.2.6d, e). This arrangement resembles the tetrahedral coordination of the fourfold bonded carbon atoms in a diamond lattice (see rod-connected diamond model as insets in Fig. 6.2.6d, e). The edges formed by cutting the bicontinuous D-surface structure model in two approximately perpendicular directions (Fig. 6.2.6f,g) not only show similar strut connected lattice structures like those of the photonic crystals (Fig. 6.2.6d,e), but also reconstruct the struts with similarly curved surfaces.

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Structural characterization

Figure 6.2.6: Ultrastructure and model of the photonic crystals in colored scales of E. imperialis. (a) OM image of a scale with two adjacent domains, yellow and green. (b) Schematic depiction of the FIB milling procedure used to expose the photonic crystals both perpendicular and parallel (gray arrows) to the scale surface. (c) Electron micrograph of the exposed photonic crystals in the two domains shown in (a). (d, e) High resolution SEM images of the edges of the photonic crystals formed by FIB milling in the yellow (d) and green domain (e) (insets: rod-connected diamond model). (f, g) Corresponding visualizations of the D-surface structure model for the yellow (f) and green domain (e).

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

Using high resolution electron micrographs of all exposed lattice planes parallel to the scale surface in various colored domains (Fig. 6.2.7), I morphometrically derived the parameters necessary to construct a D-surface structure model that recreates the natural photonic structure. I measured the distances between two ramification points in the respective <110> directions in all exposed domains. The values are close to each other and the average value is 288 ± 8 nm. Using this value, the lattice constant of this diamond-based photonic structure is determined to be 407 ± 11 nm. The thickness of the struts is similar in all domains and amounts to an average value of 98 ± 7 nm. The equal thickness of the struts and lattice constant in all domains of different scales implies that the volume fraction of the cuticular network is uniform over all scales. In order to quantify this volume fraction, I measured and determined the ratio between the diameter of the holes and the thickness of the neck of the struts in yellow domains (Fig. 6.2.7b) to be 1.61 ± 0.07. Subsequently, the D- surface structure model was adapted by fine tuning the parameter t (for details see Section 3.6) until the ratio of the same characteristic structures in the model equaled the one of the natural photonic crystal. The obtained value of t is 0.36 ± 0.03, resulting in volume fractions of 35 % cuticular network and 65 % air (± 1.5 %), respectively. The orientations of the photonic crystals in differently colored domains were approximated by fitting the structures observed in situ (SEM images in Fig. 6.2.7) with the visualizations of the different crystallographic lattice planes of the adapted D-surface structure model (model insets in Fig. 6.2.7). Upon identical visual appearance of the structure and model, the Miller indices of the lattice planes correspond to the orientations of the photonic crystals in different domains with respect to the scale surface. The normal directions of the lattice planes oriented parallel to the scale surface are close to <100> direction in green and light blue domains (Fig. 6.2.7a, d), <111> direction in yellow domains (Fig. 6.2.7b) and <110> direction in dark blue domains (Fig. 6.2.7c).

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Structural characterization

Figure 6.2.7: SEM images showing the photonic crystals in colored domains exposed parallel to the scale surface. The greyscale inserts show corresponding lattice planes of the D-surface structure model for (a) green (1 -7 40); (b) yellow (3 3 2); (c) dark blue (10 14 -3) and (d) light blue (1 2 10) domains. The insert light micrographs show the exact probed locations on the respective scales. Besides fully colored scales, I frequently identified scales with one or several transparent domains or, occasionally, even entire scales are transparent. These transparent domains appeared black when the scales were placed on the black adhesive carbon pad (e.g. upper region of the scales in the inserted optical micrographs in Fig. 6.2.8).

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

Removing the shell of transparent domains by FIB milling exposed solid cores showing periodic patterns of light and dark contrasted phases (Fig. 6.2.8a, b). The arrangement of the two phases resembles the section profiles of the photonic crystal in colored domains (e.g. compare Fig. 6.2.8a, b to Fig. 6.2.7b, a). The measured average distance between structural features in the section profiles of the transparent domains (288 ± 10 nm) is the same as the one between counterpart features in colored domains. It is possible to match the section profiles of the solid phase in the same D-surface structure model for colored domains to the patterns of the dark contrasted phase in transparent domains (compare the SEM images and the model insets in Fig. 6.2.8a, b). When they match, the complementary light contrasted phases resemble the section profiles of the air phase in colored domains (Fig. 6.2.8a, b).

Figure 6.2.8: SEM images showing the photonic crystals in transparent domains exposed parallel to the scale surface. The greyscale inserts show corresponding lattice planes of the D-surface structure model for two transparent domains: (a), (13 10 16) and (b), (1 3 9). The insert light micrographs show the exact probed locations on the respective scales.

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Chemical characterization of the transparent domains

6.3. Chemical characterization of the transparent domains

6.3.1. Energy-dispersive X-ray (EDX) spectroscopy In order to reveal the elemental composition of the second solid phase in the core of transparent domains, I performed EDX experiment on transparent and colored domains. Comparison of the spectra recorded on both FIB exposed domains (Fig. 6.3.1a, b) of the same scale (see inserted optical micrograph in Fig. 6.3.1) show significant differences in elemental composition. In contrast to colored domains (Fig. 6.3.1b), transparent domains (Fig. 6.3.1a) show an almost equal carbon to oxygen ratio and an additional, strong silicon peak.

Figure 6.3.1: (a) The exposed core of the transparent domain shows a similar lattice structure as the one of the colored domain (b), but the air is substituted by a solid phase (bright contrasted phase in (a)). Comparison of the EDX spectra of transparent (inset in (a)) and colored domain (inset in (b)) indicates that this second phase is rich in Si and O. Centre: Optical micrograph of the probed scale with a transparent and a colored domain. No elevated silicon and oxygen signals are observed from the EDX spectra (Fig. 6.3.2a) recorded on the transparent domains in an intact scale (Fig. 6.3.2b). However, the shape and location of the distribution of Si (Fig. 6.3.2c) correspond to those of the transparent domains in the scale (Fig. 6.3.2b). Such a correspondence is not detectable for the distribution of O signals (Fig. 6.3.2d).

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

Figure 6.3.2: (a) EDX spectrum recorded on the transparent domain of the intact scale shown in the optical micrograph in (b). (c, d) Elemental maps showing the distribution of Si (c) and O (d) in the scale depicted in (b).

6.3.2. Fourier transform infrared (FTIR) spectroscopy Furthermore, transmission FTIR experiments were performed to determine the chemical composition of the material in both transparent and colored domains. The spectra (Fig. 6.3.3a) obtained from a scale with a colored domain and a transparent domain (Fig. 6.3.3b) reveal the presence of two strong peaks at 1020 and 1091 cm-1 in the transparent domain which are absent in the colored domain. These two peaks are typical for Si-O stretching modes from SiO4 tetrahedra (Etchepare et al., 1974; Farmer, 1974 ; Etchepare et al., 1978; Efimov, 1995). Two peaks at 1261 cm-1 and 2964 cm-1 are of significantly higher intensity in the transparent domain compared to the colored domain. The latter can be attributed to a C-H stretching mode (Nyquist, 2001). For the former, many possibilities exist (Nyquist, 2001), though no Si-O related modes have been described in minerals in this region to our knowledge (Farmer, 1974 ). The peaks at 1650 cm-1 (protein amide I mode) (Goormaghtigh et al., 1994) and 1545 cm-1 (protein amide II mode) (Goormaghtigh et al., 1994) are of equal intensity and shape in both domains. An integration of the absorbance between 950 and 1150 cm-1 in spectra from different

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Photonic band structure calculation regions of the scale (Fig. 6.3.3c) shows that the distribution of the Si- O modes (purple region in Fig. 6.3.3c) is confined to the transparent domain (indicated by the black arrow in Fig. 6.3.3b).

Figure 6.3.3: Chemical composition of scales with transparent domains. (a) Transmission FTIR spectra of the transparent (red line) and colored (black line) domain. (b) Light micrograph of the analyzed scale (arrow: transparent domain). (c) Spatial distribution of the integrated absorbance from transmitted light of the Si-O stretching mode spectral region (blue: weak integral absorbance, purple: strong integral absorbance).

6.4. Photonic band structure calculation

To validate the structural analysis of the photonic crystals in colored scales, I compared the wavelength ranges of the observed colors to the calculated photonic band gaps using the adjusted D-surface structure model. The photonic band structures are calculated by solving Maxwell’s equations for eigenmodes in the frequency-domain using the MIT Photonic-Bands (MPB) package (Johnson and Joannopoulos, 2001). The refractive index of the cuticular network was assumed to be 1.56, which is optically determined from the scale of butterfly (Vukusic et al., 1999), and the refractive index of air is 1.00. In order to compare the optical properties between the transparent and colored domains, the photonic band structures of the photonic crystals in the transparent domains are calculated by using the same method, but replacing the refractive index of air phase by the one of SiO2 phase. Since the refractive index of most of the polymorphs of silica ranges

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

from 1.4 to 1.6 (Skinner and Appleman, 1963), I used 1.5 for the SiO2 phase. The resulting photonic band diagram (Fig. 6.4.1a) shows partial band gaps along different directions. The wavelength ranges of band gaps calculated for the photonic crystals (lattice constant: 407 nm) in colored domains (solid lines in Fig. 6.4.1a) are 527-603 nm for Γ-L <111> direction (yellow domain, Fig. 6.2.7b), 476-513 nm for Γ-X <100> direction (green and light blue domains, Fig. 6.2.7a, d) and 453-492 nm for Γ-K <110> direction (dark blue domain, Fig. 6.2.7c). I also revealed that the band gaps calculated for all investigated colored domains obtain their widest frequency ranges when the volume fraction is close to 35 %, which is the value determined for this beetle (Fig. 6.4.1b). The volume fraction of the cuticular material I calculated varies from about 8 % to 92 % (corresponding to parameter t varying from 1 to -1). In contrast, the combination of the SiO2 phase and the cuticular phase in transparent domains with differently oriented photonic structures (Fig. 6.2.8) results in very narrow partial photonic band gaps along different directions (dashed lines in Fig. 6.4.1a).

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Photonic band structure calculation

Figure 6.4.1: (a) Photonic band diagram for colored (solid lines) and transparent (dashed lines) domains calculated using the parameterized D- surface structure model. The probed locations (light micrographs) and the color range of the band gaps (color strips) for the corresponding directions are shown as inserts. (b) Band gap width as a function of varying volume fractions of cuticular material (t ∈ [1,-1]). For colored domains, the band gaps (orange: <111>; green: <100> and blue curve: <110>) obtain their widest frequency ranges at a volume fraction close to 35 % (dashed line). For transparent domains, the band gaps are very narrow (grey circle).

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

6.5. Optical characterization

6.5.1. Selective reflections of the scales The scale observed in bright field reflection mode under OM shows domains with orange, green and yellowish green vivid colorations (Fig. 6.5.1a), whereas the same individual domain of this scale shows greenish, orange and purplish hue in transmission mode, respectively (Fig. 6.5.1b). When two scales with different colorations are overlapped with each other, e.g., the upper region of one orange scale is covered with a green domain of another scale (Fig. 6.5.1c), the green and orange reflections from both scales in this overlapping region are visible and these two colorations are mixed to generate a yellowish coloration.

Figure 6.5.1: Optical micrographs of the same scale taken in reflection (a) and transmission mode (b). (c) Optical micrograph showing the mixed reflection of light from two scales with differently colored domains overlapping with each other. The reflective spectra (curves No. 1-8 in the diagram of Fig. 6.5.2) of various colored scales (showing in the bottom row of Fig. 6.5.2 with corresponding numbers) were recorded under OM. The probed regions are indicated by the black dashed circles marked on the optical micrographs of scales in Fig. 6.5.2. These regions are located in single colored domains for the spectra No. 1-6 (solid curves), whereas the spectra No. 7 and 8 (dashed curves) are recorded from regions consisting of more than three differently colored domains. The recorded spectra of all these regions show broad peaks of which the

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Optical characterization full widths at half maximum (FWHM) of reflectivity range from 73nm to 173nm (Table 6.5.1). The FWHM of the spectra for the greenish yellow or orange domains (scale No. 4-6) are broader than those for the light blue or green domains (scale No. 1, 2) with the exception of scale No. 3. The broadest peak is obtained from the sum reflection from 4 differently colored domains (including blue, green, yellow and orange) in scale No. 7. However, the spectrum of scale No. 8, which is recorded from multiple domains like in No.7, does not show a significantly broader peak compared to those of single colored domains. Compared to the simulation results, the middle points of the measured wavelength ranges at half maximum of reflectivity locate in or close to the calculated band gaps (indicated at the upper margin of the diagram in Fig. 6.5.2). The peaks of orange (No. 5, 6) and green (No. 2, 3) domains have overlaps with the calculated band gaps at corresponding <111> and <100> directions with deviations to the longer wavelength sides of those gaps. The peaks of greenish yellow (No. 4) and light blue (No. 1) domains locate mainly inside corresponding bang gaps. The measured FWHM of reflectivity are usually 30-40 nm broader than the calculated band gaps. The maximum reflectivity of investigated scales varies from 0.19 to 0.65 (Table 6.5.1). The orange scales obtain the highest reflectivity among other colored scales.

Table 6.5.1: Wavelength ranges at half maximum of the reflectivity, FWHM and maximum reflectivity of the measured spectra for various scales depicted in Fig. 6.5.2.

Wavelength ranges at half FWHM of Scale Maximum maximum of reflectivity (and reflectivity No. reflectivity their middle points) (nm) (nm) 1 460-533 (497) 73 0.40 2 479-556 (518) 77 0.29 3 443-591 (517) 148 0.19 4 508-645 (577) 137 0.34 5 532-642 (587) 110 0.59 6 538-648 (593) 110 0.65 7 434-607 (520) 173 0.25 8 486-580 (533) 94 0.31

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

Figure 6.5.2: Measured reflectivity spectra of the regions in differently colored domains (marked by dashed circles) of various scales (see optical micrographs below) at normal incidence. The calculated gap widths along different directions in Fig. 6.4.1a are indicated by the double headed arrows at the upper margin of the diagram.

6.5.2. Polarization effect Furthermore, I revealed that, depending on the polarization states of the polarizer for the incident light and the analyzer for the reflected light, the observed optical appearance of differently colored domains varies. When the position of the polarizer is fixed and the analyzer is rotated from parallel to perpendicular with respect to the polarizer, the reflection of the yellow domain continuously weakens until it disappears at cross-polarization (Fig. 6.5.3). In contrast, during the

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Optical characterization same procedure, the green domain displays very weak reflection at parallel polarization. The intensity of reflection becomes continuously stronger and is clearly visible at cross-polarization, which indicates that the polarization state of the incident light is changed after light is reflected by the scale (Fig. 6.5.3).

Figure 6.5.3: Optical micrographs showing the reflection of the same scale recorded in bright field mode and between the linear polarizer (orange arrows) and analyzer (blue arrows) set at different angles with respect to each other. In order to study this effect on reflected light and its relationship to the photonic structure, I investigated the optical responses of various green domains under different combinations of the polarization state of polarizer and analyzer together with a structural analysis of the internal structure. For a scale with a single green domain (most left image in Fig. 6.5.4a), the SEM image of the exposed photonic crystal shows that {100} lattice plane is approximately parallel to the scale surface and the <110> direction is oriented along about 45° to the 0° position of the polarizer or analyzer defined here (central image in Fig. 6.5.4a). When observed without analyzer, the reflection of the scale weakens when the angle of polarizer changes from 0° to 45° and to 90° (right three images in Fig. 6.5.4a). However, this weakening is also observed for the control experiment in which the optical micrographs are taken under the same set up for an optically isotropic silicon wafer (see Fig. 6.5.4b).

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Figure 6.5.4: (a) Optical micrograph of a scale with a single green domain recorded in bright field mode and changes of its optical appearance in dependence of the angle between the polarization state of the polarizer (orange arrows) and the <110> direction (green arrows) of the photonic crystal in this scale. The <110> direction was determined using the SEM image (center of (a)). (b) Control experiment performed on an optically isotropic silicon wafer with the same optical set-up as in (a). When the incident light is not polarized, the intensity of the reflection of the green scale gradually increases when the analyzer is rotated from 0° to 90° then decreases from 90° to 180°, and it shows maximum reflection at 90° (top row images of Fig. 6.5.5a). If the scale is rotated clockwisely for about 90°, the same trend of the change of reflection is observed during the same rotation of analyzer (bottom row images of Fig. 6.5.5a). However, if the scale is rotated clockwisely for about 45°, an inversed trend of changing the reflection is observed, i.e. the maximum reflection is obtained at an analyzer position of 0° or 180° while the minimum one is obtained at 90° (middle row images of Fig. 6.5.5a). This case, which is different from the previous two, is similar to the control experiment in which the reflection of the silicon wafer is weaker for analyzer at 90° but stronger at 0° or 180° (Fig. 6.5.5b).

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Optical characterization

Figure 6.5.5: (a) Changes of the optical appearance of the same scale depicted in Fig. 6.5.4 depending on the angle between the polarization state of the analyzer (blue arrows) and the <110> direction (green arrows) of the photonic crystal in this scale. (b) Control experiment performed on an optically isotropic silicon wafer with the same optical set-up as in (a). When the polarizer is set at 0° or 90° (top and bottom row images of Fig. 6.5.6a), the maximum reflections of the scale is obtained when the analyzer is set to be perpendicular to the polarizer (cross- polarization), and the minimum one is obtained when they are parallel with each other. The maximum reflection observed under cross- polarization is stronger when the polarizer is at 0° than the one at 90°. In contrast, when the polarizer is at 45°, the maximum reflection is obtained when the analyzer is parallel to the polarizer, whereas the minimum reflection is obtained when the analyzer is at 157.5° (middle row images of Fig. 6.5.6a). The change of the intensity of reflection from the silicon wafer following the change of the position of the analyzer in control experiment is opposite to the trend of the change

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) for the green scale in the top and bottom row images of Fig. 6.5.6a, but similar to the one in the middle row (Fig. 6.5.6b).

Figure 6.5.6: (a) Changes of the optical appearance of the same scale as depicted in Fig. 6.5.4 depending on the angle between the polarization states of the analyzer (blue arrows) and the polarizer (orange arrows). (b) Control experiment performed on an optically isotropic silicon wafer with the same optical set-up as in (a).

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Optical characterization

The polarization effect is more complicated when the <110> direction of the photonic structures in green domains (domain I and II in Fig. 6.5.7a) is not oriented along 45° to the 0° position of the polarizer or analyzer (see Fig. 6.5.7b). Domain I and II show different optical responses when observed with the same combination of the position of the polarizer and analyzer (Fig. 6.5.7c), although the intensities of the reflections of these two domains are similar when observed in bright field mode (Fig. 6.5.7a). For instance, domain I shows a stronger reflection at cross-polarization whereas the reflection of domain II is very weak with the same set-up, but is stronger when the analyzer is parallel to the polarizer. When the polarizer is set to 0°, the reflection of both domains is much stronger when the analyzer is set to 45° than the one for 135° (top row of images in Fig. 6.5.7c). The inverse trend of changing the intensity of reflection for both domains is observed when the analyzer is rotated in the same sequence, but the polarizer is set to 90° (bottom raw images in Fig. 6.5.7c). When the polarizer is set to 45°, the strongest and weakest reflection of domain I is observed when the analyzer is parallel and perpendicular to polarizer, respectively, whereas domain II shows strong reflection at cross-polarization (middle raw images in Fig. 6.5.7c). Compared to the green scale shown in Fig. 6.5.6a, the trend of changing the intensity of reflection of domain I is similar, but with the difference that the latter shows different intensity when the analyzer is set to 45° and 135°.

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

Figure 6.5.7: (a) Optical micrograph of a scale with multiple green domains recorded in bright field mode and (b) SEM image showing the different orientations of the photonic crystals inside the green domains I and II in (a). (c) Changes of the optical appearance of the green domains I and II in (a) depending on the angle between the polarization states of the analyzer (blue arrows) and the polarizer (orange arrows).

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Discussion

6.6. Discussion

6.6.1. Structural origins of the scales The structural characterization of the scales revealed that they are hierarchically organized over at least at two levels independent of the general structural hierarchy of cuticle as described in the introduction (see Fig. 2.2.1). The higher hierarchical level is the formation of a core-shell structure like in many other weevil species (Welch et al., 2007; Galusha et al., 2008; Pouya et al., 2011). At a lower level, the core has 3D periodic structures with different orientations and the shell has a layered structure in the region near the core (Fig. 6.2.4). The next lower structural level would be the biological materials that form the core and the shell. In literature, there seems to be no definitive description of the chemical composition of these materials available. In general, it is believed that each side of the scale is comprised of an outer proteinaceous epicuticle and an inner chitinous procuticle (Kinoshita and Yoshioka, 2005a). The SEM investigation show that in the shell the thicknesses of the thin layers near the core and of the outer single thick layer are in the same range of those of the layers in exocuticle and epicuticle in the elytron, respectively (Fig. 6.2.3 and Fig. 6.2.4). After polishing at an obliquely angle, the layers near the core show jagged edges which is an indication of the fibrous nature of chitinous material in the exocuticle, whereas the homogeneous appearance and the exterior position of the outer single layer indicates that it belongs to the epicuticle (Fig. 6.2.5d). Therefore, based on the information of both the thickness and structure, I suggest that the shell of the scale is formed by an outer epicuticle and several layers of inner exocuticle. Furthermore, I observed that the stems of the scales are surrounded by exocuticle which implies that they are formed by this layer and the endocuticle does not occur in the shell (Fig. 6.2.3). Because cuticle is deposited from the outer layer to the inner core, this assignment of exocuticle to the layers near the core then implies that the structured core is also part of the chitinous procuticle. Developmental studies of the formation of the scales should provide more insights on the assignment of the core to which one of the three major regions of the cuticle. However, to my knowledge, there is no such study on the formation of Entimus sales or even those of other weevil species. Most available studies were

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) focused on the formation of the scales of butterflies, which are closely related to weevil scales and often display vivid colors as well. In butterfly scales, it is known that similar inner lattice structures only start to form after the formation of external structures (lower and upper lamina, ridges and ribs) is finished (Ghiradella, 1989). Under the assumption that the formation of 3D lattice structures in the scales of E. imperialis is similar to the one of butterfly scales, this structured core is most probably formed after the formation of the shell and thus belongs to the procuticle. The observation of chitin-protein fibrils inside the struts of the core would directly prove the assignment of the core to procuticle. Fibrils have been described in the outer lamina or the inner structures during the development of the scales of moths (Overton, 1966) and butterflies (Ghiradella, 1989), but were not distinguishable in TEM micrographs of the cross section of scales from E. imperialis (Wilts et al., 2011). The observations on mechanically fractured (Fig. 6.2.2) or polished (Fig. 6.2.4) and FIB polished (Fig. 6.2.7) surfaces of the core show no indication of fibrillar structures. However, even if there are fibrils, whose center to center spacing of the constituting chitin rods is normally about 6 nm (Neville, 1975), they are most probably obscured because of the coating (2-3 nm) or the drying effect during the SEM observation and become undistinguishable. Therefore, additional characterization methods, such as TEM, X-ray analysis, and Raman spectroscopy are needed to further elucidate the chemical nature of the core and thus provide information for the determination of the refractive index of this optically active structure.

6.6.2. Photonic crystals in the core Two fundamental questions concerning the structure of the 3D biological photonic crystals are what is the basic structure of the unit cell and how the photonic crystal is modified to display different colorations. For the first question, the basic structure in the core of the scales of E. imperialis has been described before as being face- centered cubic (f.c.c.) crystal (Deparis and Vigneron, 2010) and more recently as being a D-surface structure (Wilts et al., 2011). Structural analysis here shows that the fourfold connected cuticular struts form a structure resembling the rod-connected diamond structure (Fig. 6.2.6d, e). The D-surface structure is a better fitted model since it more

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Discussion accurately recreated the curvature at the connecting point between two struts (Fig. 6.2.6f, g). Whether this D-surface model is valid for all investigated differently colored domains is not immediately obvious, since the appearance of the exposed photonic structure in different domains can be very different (Fig. 6.2.7). This observation raises the second question: is the basic structure everywhere the same in differently colored domains? Theoretically, different colors can be generated by varying the volume fraction of the cuticular material, the lattice constant and the orientation of the crystal with respect to the scale surface. The results show that lattice constants and the thicknesses of the struts are equal among differently colored domains, which implies that the volume fraction and thus the parameter t are constant. Therefore the parameter t that was determined quantitatively for a yellow domain (Fig. 6.2.7b) can be used to adapt the D-surface model for the photonic crystals in all domains. By adjusting visualizations of the D-surface model to match the appearance of the section planes, it is able to derive the orientation of the photonic crystal in each domain. Direct comparison of the model cut along the same lattice plane as the photonic structure shows remarkable structural resemblance (Fig. 6.2.7). Therefore, based on the structural analysis, I revealed that the photonic crystals in differently colored domains have the same basic D-surface structure and only the orientation of them varies among different domains. Whether the colorations of different domains correspond to the photonic band gaps for the derived orientations in corresponding domains will be discussed in Section 6.6.3. Transparent scales have been observed before in the weevil Pachyrhynchus argus (Parker et al., 2003) and have been explained by the absence of an inner opal-type photonic crystals as found in its colored scales. The results here show that transparent scales of E. imperialis contain a 3D structure that resembles the photonic crystal in colored scales (Fig. 6.2.8 and Fig. 6.2.9). Since the D-surface model can be oriented and sectioned to exactly match the section profiles observed in the investigated transparent domains (Fig. 6.2.8), one can assume that the structure of the photonic crystal is similar in transparent and colored domains. Thereby, the dark contrasted features correspond to the solid phase and the light contrasted features to the air phase in colored domain (Fig. 6.2.8). This implies that in the

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) transparent domains the air is substituted by another solid phase. EDX analysis on the transparent domains exposed by FIB milling unambiguously shows that they are silicon and oxygen rich compared to colored domains (Fig. 6.3.1). However, in intact scales the transparent domains are covered by the shell. Thus, the detected Si and O signals are by far not as elevated as those obtained from exposed transparent domains (Fig. 6.3.2a). Nevertheless, elemental mapping of scales with both types of domains reveals that Si is located exclusively in the transparent domains (compare Fig. 6.3.2b and c). The absence of visible contrast of the O signal between intact transparent and color domains (Fig. 6.3.2d) can be explained by the lower energy of the characteristic X-rays emitted by O in comparison to Si, which causes them to be absorbed more easily by the shell. Under the assumption that the composition of the cuticular phase in both types of domains does not differ, the elevated silicon and oxygen signals should originate from the solid second phase present in the transparent domains. This case would be suitable to explain the unusually high electron optical contrast between the two phases observed in the transparent domain upon SEM investigation, which should normally show almost no contrast because of the flat surface resulting from FIB preparation. The samples were observed using an in-lens detector, which can detect not only secondary electrons which are responsible for topological contrast but also back scattered electrons which generate characteristic atomic number contrast. Due to the fact that the atomic number of silicon is more than two times higher than that of carbon, the carbon rich cuticular network appears dark contrasted and the silicon rich phase appears light contrasted. Additionally, FTIR spectroscopy revealed high absorbance in the Si-O stretching modes region in the transparent domain (Fig. 6.3.3). The similarity of the amide mode shown in the spectrum of the colored and transparent domain implies equal protein density and structure in both domains. Together with the structural resemblance, it is reasonable to assume that the cuticular network is similar in both domains. Therefore, the additional CH peak indicates the presence of additional organic material besides silicon oxide species in the transparent domain. Although the existence of Si in the scale of this weevil seems to be surprising, together with silicon, many inorganic elements can be found in the cuticle of insects (Richards, 1956; Rockstein, 1974). The

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Discussion high abundance of silicon and oxygen elements in the second phase of the transparent domains indicates the existence of a type of silicon oxide. No significant amounts of other elements typically present in silicate minerals, such as Na, K, Ca or Al have been found. In addition, the IR spectra show the presence of Si-O modes typical for the most abundant four-fold coordinated silicon in SiO2 (Etchepare et al., 1974; Etchepare et al., 1978). Other forms of SiO2 exist, but are extremely rare (Lyon, 1962).

At present, it is not known whether the SiO2 present in transparent scales is originating from the beetles themselves or the result of some contamination process that occurred in vivo or post mortem during storage in museum collections. Concerning post mortem contamination, I traced potential scenarios, e.g. conservation method, preservation agents etc. to the best of my knowledge without being able to find evidence for artificial SiO2 sources. However, the fact that Parker et al. (2003) found transparent scales in another weevil species (P. argus) as well indicates that they are formed by the animals themselves. Further, the FTIR spectra of the SiO2 modes observed here closely resemble those observed in biomineralised silica and show substantial differences to man-made SiO2 where the peaks at 1090 and 1020 cm-1 are usually not of equal intensity (Sandford, 2003). A physiological mechanism for the incorporation of SiO2 into the photonic crystals inside the scales of E. imperialis has not been described yet, and given the existence of such a mechanism, it would still be important whether this happens during the development of the scales or later. If SiO2 is actively metabolized by the beetles, one would have to raise the question what purpose this would serve. At the current state, I could only speculate about the biological role of such an adaptation, but this is certainly a very interesting topic for further investigation.

6.6.3. Optical properties of the scales The scales of E. imperialis are strongly iridescent which can be observed already with bare eyes. This is different from the scales of previously reported weevils like Lamprocyphus augustus, which shows macroscopically near angle-independent green coloration (Galusha et al., 2008), or the uniformly orange scales of

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

Pachyrrhynchus congestus pavonius (Welch et al., 2007). Earlier, the iridescence of the scales of E. imperialis has been explained by assuming the photonic crystals in individual scales to be single crystals, i.e. each scale shows its own color (Deparis and Vigneron, 2010). Wilts et al. (2011) and my observations show that, just like in the other weevil species mentioned above, most of the scales have several well defined differently colored domains (Fig. 6.1.1c, d and Fig. 6.5.1). However, although the size of entire scales is similar, individual domains in scales of L. augustus and P. congestus are much smaller than those of E. imperialis. Here, the domains are large enough to serve as a single photonic crystal to reflect different colors when observed from different angles whereas the tiny microdomains of the other species cause a color stirring effect that prevents observing iridescence. However, similar to color stirring, a color mixing effect is also observed for the scales of E. imperialis when two colored domains are overlapped with each other. The light reflected by the top and bottom photonic crystals is mixed and generate a third color which should cover a broader wavelength range indicated by the resulting strong metallic golden color (Fig. 6.5.1c). The concept of overlapping 1D photonic crystals with different layer thickness to produce broadband reflectors has been described in the cuticle of beetles (Parker et al., 1998). Here this concept is extended to 3D photonic crystals with different orientations which have potential of further modification and functionalization. Since 3D photonic crystals that work in the visible spectral range are difficult to synthesize, the single biological photonic crystals in different domains provide an opportunity to study the optical properties of such structures. The experimental results show that it is the direction close to <111> parallel to the surface normal of the orange domains, <100> parallel to the one of green or light blue domains and <110> parallel to the one of dark blue domains (Fig. 6.2.7). The simulation results show that the wavelength ranges of the photonic band gaps calculated using the D-surface model in these three directions (colored strips in Fig. 6.4.1a) show good agreement with the observed colors in the domains with corresponding orientations (optical micrographs in Fig. 6.4.1a). The reflective spectra also show that the peak positions of the probed individual domains coincide with the locations of the band gaps of corresponding

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Discussion directions (Fig. 6.5.2). This result confirmed the conclusion from the structural analysis that the orientation is responsible for the reflection of light with different wavelengths in different domains. It also proved that the D-surface model can be considered to be an accurate numerical recreation of the photonic crystals in scales of E. imperialis. Most studies up to date only correlated the recorded reflection peak positions with the simulated band gap positions to validate their models abstracted from structural analysis. However, besides the peak positions, the spectra also provide information on the width and reflectivity of individual reflection peaks. The measured spectra show that the FWHM of the peaks for orange or yellow domains are broader than those of the green or blue domains (Fig. 6.5.2). This difference in peak width is reflected by the theoretically predicted larger band gap width of orange domains compared to the one of green or blue domains (Fig. 6.4.1a). The larger band gap of orange domains indicates higher photonic interaction strength (ψ = ∆ω/ωm), and thus less lattice planes needed to build up a Bragg reflection (Bragg length LB= 2 d /πψ) (for details see Section 2.3.1). This Bragg attenuation length for orange domains calculated based on the band gap size equals to the thickness of about 5 times the interplanar distance in <111> direction (Table 6.6.1). The number of lattice planes along <111> direction is about 16 which is estimated by dividing the thickness of the core (3.7µm) by the interplanar distance (234nm). Since the incident light experiences an exponential decay in the band gap, the predicted theoretical reflectivity for orange domains is 1 = 0.96 (Huisman et al., 2011) which is about 30 % higher than−16⁄ 5the measured value (Table 6.5.1). This reduction of measured− 푒 reflectivity can be explained by the losses caused by the scattering by the shell, the deviation of the surface normal of the scales from the incident light or the theoretical lattice planes are not perfectly parallel to the scale surface. The Bragg lengths calculated for the domains with <100> and <110> directions parallel to the surface normal are 9d<100> and 8d<110>, respectively, which are larger than the one of the orange domains (Table 6.6.1). The numbers of lattice planes along these two directions are close to the number for orange domains. Therefore the predicted reflectivity of the domains with these two orientations is smaller than the one of the orange domains (Table 6.6.1). The experimental results (Fig. 6.5.2) show that the - 130 -

Chapter 6 | Three-dimensional Photonic Crystals (Weevils) reflectivity of the probed two orange domains is higher than that of domains with other colors, which agrees well with the conclusion from the theoretical prediction. Therefore, domains with photonic crystals which can open up larger band gaps have higher reflectivity, when the number of the lattice planes is similar in photonic crystals in different domains. Band gap calculations on the D-surface structure model with volume fractions of the cuticular material varying from about 8 % to 92 % (corresponding to t varying from 1 to -1) show that all investigated differently oriented domains obtain their largest band gaps when the volume fraction is close to the value observed in E. imperialis (35 %) (Fig. 6.4.1b). This results implies that, by varying the volume fraction of the cuticular network, the three typical colored (orange, green, blue) domains all obtain the highest possible reflectivity for the given materials and structural configuration.

Table 6.6.1: Estimation of the Bragg length and reflectivity of the photonic crystals along different crystallographic directions.

N : A Interplanar N : Bragg B Gap distance Number Direction length Reflectivity: size ψ= d (nm) of planes LB in 1 ∆ω/ωm where along number 퐵 퐴 a = 407nm −푁 ⁄푁 of d − 푒 3 <111> 0.134 5 = 234 16 0.96 3 √ 푎 <100> 0.073 9 = 204 18 0.86 2 푎2 <110> 0.082 8 = 192 19 0.91 3 √ 푎 It is widely accepted that diamond based structures are best suited for photonic crystals in terms of their ability to open up large complete photonic band gaps (Maldovan and Thomas, 2004). As an example, the rod-connected diamond structure is able to open the largest band gaps of any structure studied up to now (Chan et al., 1991; Maldovan and Thomas, 2004). Moreover, it requires the smallest refractive index contrast (slightly less than 1.9) for the onset of a complete photonic band gap (Chan et al., 1991). Given that the refractive index contrast

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Discussion of biological photonic crystals is normally smaller (around 1.5) due to the limited choice of materials living organisms have at their disposal, the diamond-based structure is more efficient to open up photonic band gaps than other structures. Theoretical work has shown before that the volume fraction of the dielectric structure for the onset of a complete photonic band gap at the lowest possible refractive index contrast is 33 % for rod-connected diamond structures (Chan et al., 1991) and 34 % for D-surface structures (Maldovan et al., 2002). Interestingly, the volume fraction of the cuticular material in the photonic crystal of E. imperialis is 35 %, which is very close to both the values pointed out above. Therefore the results show that E. imperialis develops a optimized basic structure with the optimized structural parameter in the colored domains to open up largest possible band gaps, the same strategy physicists applied to open up complete band gaps, in order to obtain highest possible reflectivity for structural coloration. For the transparent domains, I revealed that they form the same D- surface structure like in the colored domains, but the air phase is substituted by a second solid silica phase. It is known that the refractive index of most of the polymorphs of the silica ranges from 1.4 to 1.6 (Skinner and Appleman, 1963), which is very close to the most accepted refractive index of the cuticular material (1.56). In contrast to the large band gaps obtained in the colored domains, this combination of the silica phase and cuticular phase can only open up very narrow photonic band gaps due to the very small refractive index contrast between them (Fig. 6.4.1). These narrow gaps lead to weak reflection while most of the light transmits through the scale. This weak reflection is sensitive to scattering. As a result, the domain consisting of these two phases appears transparent and dull to the eye. Regardless of the provenience of SiO2 in the transparent domains of the scales and its potential functions, the principle of replacing one phase of a photonic crystal by another one with different refractive index is a highly interesting concept from a materials science point of view, since it is a natural example for tunable photonic crystals.

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils)

6.6.4. Polarization effect Polarization effects have been described for the structural coloration of the scales of the butterfly species Parides sesostris (Vukusic and Sambles, 2001) and Callophrys rubi (Saba et al., 2011). The photonic crystals in the scales of these two butterfly species share the same triply-periodic gyroid structure, a body-centered cubic (b.c.c.) structure, with different structural parameters (Michielsen and Stavenga, 2008). The chiral network of the gyroid structure leads to selective reflection of circularly polarized light in <100> direction in the scales of C. rubi (Saba et al., 2011), which share the same mechanism to open up the polarization band gaps as described for spiral photonic crystals (Lee and Chan, 2005). Observation and theoretical prediction of the change of the polarization state of linearly polarized light have been reported for the synthetic f.c.c. opal films (Romanov et al., 2010; Wolff et al., 2011). Both reports show that there exists a strong cross-polarized transmittance (s-polarized light in, p-polarized light out) when the s-polarized light is incident in a direction of about 50° with respect to the surface normal (<111> direction) of the opal film in the Γ-L-W plane of the first Brillouin zone. This effect is very sensitive to the variations of the plane of incidence (Wolff et al., 2011). This seemingly unusual birefringence- like polarization transfer effect demonstrates that the statement of optical isotropy for photonic crystals with cubic symmetry only holds in the limit of an effective material. When the relevant feature size of the photonic crystals is similar to the wavelength of light, they cannot be modeled as an effective material, at least as soon as polarization is involved (Wolff et al., 2011). For the weevil E. imperialis, I often observed bright reflections under cross-polarization for the green domains in the scales, whose <100> direction is close to parallel with the surface normal. This observation indicates a polarization transfer effect for the reflected light from the photonic crystals with D-surface structure, similar to the effect that occurs in the synthetic opal films for the transmission of light mentioned above. Whether the polarization transfer effect I observed is sensitive to the plane of incidence is unknown, since an infinite number of planes of incidence exists in the light cone incident from the objective of the microscope. However, from various

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Discussion observations, I found that this effect is strongly sensitive to the plane of polarization, i.e. the angle between the electric field vector (E vector) of the linearly polarized light and the <110> direction in the {100} lattice plane (this plane is parallel to the scale surface of green domains). Changing this angle by rotating the plane of polarization of the incident light (Fig. 6.5.6a), thus the direction of E vector, or probing the photonic crystals in two neighboring domains with different <110> directions (Fig. 6.5.7) both lead to different intensity of cross-polarized reflections when the other parameters remain constant. The trend of the variation of this intensity provides the information to estimate the direction of the E vector of reflected light. For clarity, the rod-connected diamond structure is used as a simplified model for the photonic structures in the scales (Fig. 6.6.1) to show the angular relationship between the direction of the E vector of incident light (Ein, dashed line arrow) and the [110] direction of the photonic crystal, and also the corresponding direction of the E vector of reflected light (Eout, solid line arrow). The photonic crystal in the single domain scale shown in Fig. 6.5.6a is oriented in a “standard” way, i.e. a nearly symmetrical position with respect to the sample coordinate (angle between [110] and X axis is 48°), thus also the investigated planes of polarization. When the Ein vector is oriented approximately 45° to the [110] direction, the polarization transfer effect is strong (top and bottom row of images in Fig. 6.5.6a), thus the Eout vector is rotated by 90° with respect to the Ein vector (inset I, II in Fig. 6.6.1a). In contrast, this effect is much weaker when the angle between the Ein vector and [110] direction is about 90° (inset III in Fig. 6.6.1a), since the variation of the reflectivity of the scales follows the same trend for the one of the silicon wafer (optically isotropic) in the control experiment (compare the middle row of images in Fig. 6.5.6a and b). However, the polarization transfer effect still exists to a certain extent in this case, indicated by the observation that the weakest reflection does not occur at cross-polarization, but at analyzer set to 157.5° (last image in the middle row of Fig. 6.5.6a). Therefore the Eout vector is about 22.5° clockwisely deviated from the Ein vector (inset III in Fig. 6.6.1a). In another scale with two differently oriented green domains (Fig. 6.5.7a), domain I shows a strong polarization transfer effect when the

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) plane of polarization is set to 0° or 90° (top and bottom row of images in Fig. 6.5.7c), which is similar to the optical response of the single domain scale in Fig. 6.5.6a. This similarity is most probably because the direction of [110] in domain I is closer to the [110] direction (14°) than the [100] one (31°) of the single domain scale (compare Fig. 6.6.1a and b), leading to a similar angular relationship between the Ein vector and [110] directions for these two domains. In contrast, the [110] direction of domain II in Fig. 6.5.7b is closer to the [100] direction (13°) of the single domain scale (compare Fig. 6.6.1a and c). Therefore, the strong polarization transfer effect for domain II is observed when the plane of polarization is set to 45° (fourth image in the middle row of Fig. 6.5.7c), instead of 0° or 90° for domain I. The optical response of domain I and II to the linearly polarized light again indicates that strong polarization transfer effects occur when the plane of polarization (or the Ein vector) is close to 45° oriented towards the <110> direction, i.e. parallel to the {100} lattice plane of the photonic structure in the green domains. Another notable optical response of the domain I and II is that the reflectivity of both domains at an analyzer setting of 45° is always different from that at 135° even though the plane of polarization for the polarizer is the same (Fig. 6.5.7c), which is different from the observation of the single domain scale shown in Fig. 6.5.6a. For instance, the domain I shows a strong reflection when the analyzer is set to 45°, which is similar to the one at 90°, but a much weaker reflection at 135° (top row images in Fig. 6.5.7c). Therefore, the Eout vector in this case is not exactly perpendicular to the Ein vector but is deviated from this orthogonal position anticlockwisely for less than 45° (inset I in Fig. 6.6.1b). This deviation is a result of the misorientation between the photonic crystal in domain I and the one with a more symmetrical orientation in the single domain scale in Fig. 6.5.6a. This observation again shows that the optical response of the green domains is sensitive to the plane of polarization. To complete, the reflection at cross-polarization for the polarizer set to 90° is weaker than the one of 0° (bottom and top rows in Fig. 6.5.6a and Fig. 6.5.7c, respectively) due to the optics of the microscope (see the control experiment in Fig. 6.5.4b and 6.5.5b).

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Discussion

Figure 6.6.1: (a, b, c) Rod-connected diamond structure model showing the angular relationship between the direction of the electric field vector of incident light (Ein, dashed line arrow), reflected light (Eout, solid line arrow) and [110] directions of the photonic crystals in (a) single green domain in Fig. 6.5.6 and (b, c) green domains I and II in Fig. 6.5.7. (d) Model showing the plane of polarization parallel to lattice planes with ({110}) or without ({100}) mirror symmetry. The reason why the polarization transfer effect occurs when the plane of polarization is parallel to the {100} lattice plane but not the {110} one of the diamond based structure is because microscopically this structure has no mirror symmetry about the {100} plane, but about the {110} plane (Fig. 6.6.1d). This break of the symmetry about {100} lattice plane is not immediately visible when one observes the

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) exposed {100} facets in the scales (Fig. 6.5.4a, Fig. 6.5.7b). However, this break of symmetry becomes obvious if one marks the struts at different depth white and black (Fig. 6.6.1). While the struts appear mirror symmetric to each other in 2D with the {100} plane as the plane of symmetry (Fig. 6.6.1a, b, c), 3D inspection shows that they actually are not mirror symmetric to each other (Fig. 6.6.1d). This break of symmetry is probed by the light with the wavelength similar to the lattice constant of the D-surface structure in the scales, which is most probably the reason for the polarization transfer effect. This finding echoes the polarization transfer effect observed by Wolff et al. (2011) for synthetic opal films, but is different from the latter case in several aspects. First of all, the polarization transfer effect observed here exists in the reflected light, i.e. inside the photonic band gap, whereas the one observed by Wolff et al. exists in the transmitted light, i.e. beyond the band gap. Since the position of the band gaps of a photonic crystal is relatively easier to be designed compared to the region beyond the band gaps, the polarization transfer effect observed here can be better controlled in potential optical applications. Second, the polarization transfer effect of the synthetic opal film is observed when the {111} lattice plane faces to the light of incidence, but no such effect is observed for the orange domains in the scales whose {111} plane is also close to parallel to the reflecting surface. However, it is worth to note that the optical configuration for one to observe the polarization transfer effect in synthetic opal film is restricted to a specific plane (Γ-L-W plane) and angle (around 50°) of incidence in transmission, and does not occur in the Γ-L <111> fundamental band gap. In the case of D-surface structure, the orange domain is observed in reflection mode, the plane of incidence is not defined and the angle of incidence spans over the light cone formed by the objective. Therefore it is difficult to compare the optical properties of the {111} planes of these two systems and speculate the reason for the difference between these two systems. Third, the polarization transfer effect for the green domains is already visible when the angular range of incident light from the objective (NA = 0.3) is less than 17°. It is difficult to experimentally prove that this effect also occurs at strictly normal incidence, because the small size of the scales always requires them to be observed under a microscope. Nevertheless, this angle (17°) is already much smaller than the angle

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Discussion

(50°) required for the observation of polarization transfer effects in opal films. Therefore, for both theoretical study and practical application of this polarization transfer effect, the {100} family of lattice planes of diamond based structures is a more suitable system compared to the {111} family in the opal film. The above explanation for the polarization effect of scales is based on the assumption that the constituting materials of the rod-connected diamond structure model are optically isotropic. Since the photonic crystals in the scales are formed by cuticular materials which are possible to be optically anisotropic, this polarization effect of the whole photonic crystal can also originate from the inherent optical anisotropy of its constituents. In order to check whether this inherent anisotropy exists in scales, IR absorption spectra of scales were measured between crossed polarizers. The recorded intensity was, however, totally extinct between crossed polarizers. Hence, there is no polarization effect as in visible spectral range, which implies that the cuticular materials forming the photonic crystals are most probably optically isotropic. However, the structuring of the photonic crystal at a larger length scale than the molecular level may homogenize the optical anisotropy of its constituents that hinder the polarization effect to be observed in IR spectral range. Therefore, it is difficult to rule out the possible contribution from the optical anisotropy of the cuticular materials to the observed polarization effect at the current state. However, no hints to its importance are observed. Another explanation one may expect for this polarization transfer effect is that the D-surface structure in the weevil scales also decomposes the linearly polarized light into two circularly polarized lights with different handedness and selectively reflects one of them, similar to the mechanism of the polarization effect observed for the gyroid structure in the scales of some species of butterfly. This explanation is supported by the fact that not only the gyroid structure, but also the diamond-based structure, like in our case, can be formed by arrangements of spiral structures along <100> directions (Chutinan and Noda, 1998). However, in the diamond-based structure, two sets of spiral structures with opposite handedness coexist (indicated by black and white spirals in the rod-connected diamond model in Fig. 6.6.2). Thus the diamond-based structure as a whole is not a chiral

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) structure. Optically, if the D-surface structure selectively reflects circularly polarized light, one should observe similar intensity of reflected light independently from the polarization state of the analyzer when the one of the polarizer is fixed. This is contradictory to our observation that the green domains show much stronger reflection at cross-polarization compared to other configurations of polarizer and analyzer (Fig. 6.5.6a and Fig. 6.5.7c). Therefore the photonic crystal in the orientation present in green domains acts more like a linearly birefringent material rather than a circularly one. To complete, although the shell of the scales may have a helicoidal structure like the one in the exocuticle of the Scarab beetles (see Chapter 5), it does not cause the polarization transfer effect observed here. This conclusion is supported by the observation that removal of the shell does not alter the optical response of the scales (Fig. 6.2.5) and the polarization transfer effect does not occur in all colored domains which, however, are all covered by the shell (Fig. 6.5.3).

Figure 6.6.2: Rod-connected diamond structure model showing the coexistence of two sets of square-shaped spiral structure with opposite handedness (indicated by black and white spirals) resulting in a non-chiral structure.

6.7. Summary

The colors of the scales of the weevil E. imperialis are generated by photonic crystals with a bicontinuous cubic structure based on the diamond lattice structure (D-surface structure). These three-

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Summary dimensional photonic crystals form the core of the scales which is covered by a continuous shell. The shell of the scales is formed by an external epicuticular layer and an inner exocuticular layer close to the core. Since the core is secreted after the formation of the shell, it should consequently be part of the chitin/protein based procuticle. The photonic crystals in differently colored domains have the same lattice constant and volume fraction of cuticular materials. The different crystallographic orientations of these photonic crystals with respect to the surface of the scales are responsible for the different colors displayed by these domains. I revealed that the volume fraction of the cuticular phase of the photonic crystals is optimized to open up the widest photonic band gaps possible for the three orientations corresponding to the three most frequently observed colors (yellow, green and blue). Since wider band gaps lead to higher reflectivity of the photonic structure, the coloration of the scales appears very bright. I discovered that in some domains and even entire scales the air phase inside the photonic structure is substituted by a second solid SiO2 phase, although the basic structural parameters are the same as in colored domains. The refractive index contrast between the cuticular phase and the SiO2 phase is very low and these domains and scales appear transparent. The transparency can be explained by the resulting very narrow band gaps that allow for transmission of most of the light and only a small portion is reflected. These findings inspired the fabrication of high- temperature resistant SiO2-based 3D photonic crystals with tunable optical properties by biotemplating the photonic crystals in colored scales (Van Opdenbosch et al., 2012). The D-surface structure in the scales of E. imperialis displays an unusual orientation dependent polarization effect as indicated by the bright reflection observed for green domains between crossed polarizers. This effect is not present for yellow or orange domains. For green domains, this polarization effect only occurs when the plane of polarization is parallel or close to parallel to those {100} lattice planes that are perpendicular to the surface of the scales. The reason for this sensitivity to the plane of polarization is that the D-surface structure has no mirror symmetry about the {100} plane. This break of

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Chapter 6 | Three-dimensional Photonic Crystals (Weevils) symmetry causes the structure to be optically anisotropic towards light within the structure specific wavelength range. The orientation dependent polarization effect of the D-surface structure in the scales of E. imperialis is certainly worth further investigation due to its potential for applications such as devices for polarization detection or signaling. Although all evidence indicates that this effect is structure related, it is still difficult at the current state to rule out the existence of a possible intrinsic birefringence of the materials constituting the structure which may also contribute to this polarization effect. Further structural and chemical characterization using methods such as TEM, X-ray analysis and Raman spectroscopy can be carried out to illustrate the material aspects of the photonic structure. The synthesis of a respective structure of large size by advanced synthesis methods such as direct laser writing and a thorough characterization of its polarization transfer characteristics would provide the understanding necessary for potential applications.

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Chapter 7 | Conclusions

Chapter 7. Conclusions

The main objective of this study was to investigate the relations between structure and the optical properties of the cuticular photonic crystals of different beetle species belonging to three different systematic groups. Using an interdisciplinary approach, I investigated the cuticle forming selected exoskeletal parts to reveal where and how the material is modified to form color producing photonic crystals. For each group of beetles, I clarified the structural origin of the photonic crystals, determined their microstructure and, as far as possible, their composition experimentally. Then, I characterized the optical properties of these photonic structures both experimentally and theoretically to reveal their structure-property-relations. The structural models for simulations are generated using the morphometrically derived parameters from the investigated beetles. This work’s contributions to the field of biological photonic structures are summarized in the following paragraphs.

• All three groups of beetles obtain their colors by modifying their cuticular structures. The modifications occur on different levels of the structural hierarchy and in different degrees of complexity, but without compromising the major function of the cuticle. The investigated Ground beetle and Scarab beetle species have integrated the optical functions into their epicuticle and exocuticle, respectively. The Ground beetles modify their epicuticle by forming a multilayer structure, whose major function is to serve as an environmental barrier, while the Scarab beetles fully integrate optical and mechanical properties in their exocuticle by using the optical anisotropy of the constituting chitin/protein fibers and their structural arrangement. In terms of materials science, these combinations of different properties in one composite material make the cuticle of Carabidae and Scarabeidae a multifunctional material. In the weevil species, three-dimensional (3D) photonic crystals with a diamond based bicontinuous cubic

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Conclusions

structure (D-surface structure) are formed in the scales which are outgrowths of the cuticle. Here, the optical properties are functionally isolated from the major function of the cuticle which is to serve as exoskeleton.

• The set of biological constituents available to the beetles to form photonic crystals are limited. Therefore, structuring plays an important role to obtain the optical properties corresponding to the requirements imposed by ecophysiological strains acting on the individual species. This is manifested in the structural optimization observed for the D-surface structure in the scales of E. imperialis, both in terms of the basic structure and the volume fractions of the constituting phases. This leads to the widest photonic band gaps possible along different directions and thus high reflectivity for different domains in the scales. Other optical properties of the D-surface structure such as the wavelength range of the reflected light and the polarization effect also depend on the structural parameters (orientation) of these 3D photonic crystals. In the case of the two Ground beetle species, the structuring also has a significant influence on the optical properties of their less complex one-dimensional photonic crystals where the thicknesses of the outermost layers of both species are optimized to reduce the reflectivity. In the case of the investigated Scarab beetle species, the rotation angles of superimposed chitin/protein fiber planes determine the stacking height of the helicoidal structure and thus the wavelength range of the reflected light. In addition, this structural arrangement is optically unique for its ability to reflect circularly polarized light. Other Scarab beetle species evolved an additional level of structuring in their exocuticle by modifying their pore canal system to vertical cuticular rods pervading the cuticle. This additional structuring results in unusual optical properties, such as the unsaturated reflection at normal incidence of light, although the ability to reflect circularly polarized light is lost.

• Besides the structuring of the cuticle, some of the investigated beetles also modify the chemical composition of the constituting phases of the photonic crystals to obtain the

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Chapter 7 | Conclusions

required optical properties. In a part of the scales of the weevil, the air phase in the photonic crystals is replaced by silica to form transparent domains. In both Ground beetle species, the absorptive pigment melanin exists in the multilayer reflectors, which reduces the reflectivity of the cuticle. Understanding the structure-property-relations and design principles of these biological structures can provide inspirations for designing novel optical materials. For instance, the orientation dependent polarization effect discovered in the scales of E. imperialis demonstrates that the polarization properties of an optical material can be tailored and controlled by structuring the architecture inside the photonic crystals. Once fully understood, this mechanism could be incorporated into synthetic photonic structures. The design principles of cuticular photonic crystals can provide inspirations for the development of multifunctional synthetic materials where different properties are incorporated and in a tailored equilibrium. For instance, mechanical properties of synthetic photonic crystals become especially important when applications such as coating, textile fabrication, and stress sensing are under consideration. Organic constituents like some polymers are suitable for optical applications requiring ductility of the photonic materials, but have the disadvantage of their generally low refractive indices. How to enhance the optical properties of polymer based photonic crystals through modifications of the constituting phases is therefore a promising field for future investigations. The incorporation of pigments as observed with melanin in the epicuticular multilayer structures of the Ground beetles may provide a solution for this problem. Not only the mechanical properties of the constituting materials, but also the higher order structural parameters of the photonic crystals have an influence on the overall mechanical properties of the system. Within the Insecta, butterflies and weevils like E. imperialis have evolved photonic crystals with Gyroid and D-surface structure in their scales, respectively. Whether the choice of structure type during evolution was influenced by mechanical constraints is unknown, since little is known about the mechanical behavior of these bicontinuous cubic structures. Therefore, it is interesting both for biologists and

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Conclusions materials scientists to systematically investigate the mechanical and optical properties of the photonic crystals based on bicontinuous cubic structures. The obtained knowledge on how the optical properties of these differently structured photonic crystals respond to external stresses should provide the design criteria for biomimetic photonic crystals that react to deformation with a quantifiable spectral shift. Such mechanochromic sensors could sense and quantify local deformation in a simple and straightforward manner.

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Appendix

Appendix

Example script for photonic band structure calculation The script (so-called control file for “MIT Photonic Bands” package) for calculating the photonic band gap structure shown in Fig. 6.4.1a: (set-param! resolution 16) (set-param! mesh-size 16) (set-param! num-bands 12)

; A D-surface (diamond) structure of dielectric network in air, on a fcc lattice

(set! geometry-lattice (make lattice (basis-size (sqrt 0.5) (sqrt 0.5) (sqrt 0.5)) (basis1 0 1 1) (basis2 1 0 1) (basis3 1 1 0)))

; Corners of the irreducible Brillouin zone for the fcc lattice, in a canonical order: (set! k-points (interpolate 4 (list (vector3 0 0.5 0.5) ; X (vector3 0 0.625 0.375) ; U (vector3 0 0.5 0) ; L (vector3 0 0 0) ; Gamma (vector3 0 0.5 0.5) ; X (vector3 0.25 0.75 0.5) ; W (vector3 0.375 0.75 0.375) ; K (vector3 0 0 0 )))) ; Gamma

; define a couple of parameters (which can be set from the command- line) (define-param epsc 2.43) ; the dielectric of cuticular material

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Appendix

(define-param isoval 0.36) ; isovalue (t) to choose volume fraction (f) ; t=0 - f=50 %, t=0.36 - f=35.2 %

(define pi (* 4 (atan 1))) ; 3.14159...

; The Epsilon function that returns the dielectric constant as a function of position ; returns epsc if the value of the level-set function F > isoval, or else returns 1 ; F(x,y,z) = cos(2*pi*z).sin(2*pi*x+2*pi*y)+sin(2*pi*z).cos(2*pi*x- 2*pi*y)

(define (eps-func p-lattice) (let* ((p (lattice->cartesian p-lattice)) (x (vector3-x p)) (y (vector3-y p)) (z (vector3-z p)))

(if (> (+ (* (cos(* 2 pi z)) (sin (+ (* 2 pi x) (* 2 pi y)))) (* (sin(* 2 pi z)) (cos (- (* 2 pi x) (* 2 pi y))))) isoval) (make dielectric (epsilon epsc)) (make dielectric (epsilon 1))) ))

; make it the default material (set! default-material (make material-function (material-func eps- func)))

(run)

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Abstract

Abstract

Photonic crystals are optically active materials that can inhibit the propagation of light within certain frequency ranges. This type of material has shown many new and exciting optical effects which have led to considerable advances in technology. Investigating the design of the periodic structures of photonic crystals is one of the essential research fields aiming at optimized optical properties for technical applications. In nature, many living organisms have evolved biological photonic crystals which are able to generate vivid iridescent colors. These biological model systems, especially the photonic structures formed by the cuticle of insects, have already been shown to be a rich source of inspiration for the design and development of synthetic optical materials.

In this study, I investigated the relations of structure and optical properties of the cuticular photonic crystals of five beetle species from three different systematic groups with the goal to evaluate their design principles. A large variety of methods for microscopic investigations and chemical analysis were applied to characterize the microstructure and composition of these photonic crystals experimentally. Their optical properties were both experimentally measured and theoretically characterized based on models derived from the structural analysis.

The results show that the investigated species of Ground beetles and Scarab beetles have evolved multilayer structures and helicoidal structures in different regions of their cuticles: epicuticle and exocuticle, respectively. The weevil species has formed three-dimensional photonic crystals based on a D-surface structure (diamond) in scales which are outgrowths of the cuticle. Through modifications of structure and chemical composition of these basic structures, the investigated beetles obtain optical properties that are optimized to meet the imposed ecophysiological strains. In some cases, unusual and unexpected optical effects were observed. The principles of both the basic structuring of these photonic crystals and the additional modifications provide us inspirations for the design of synthetic composite materials with not only novel optical properties, but also combinations with other physical properties in a balanced equilibrium.

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Zusammenfassung

Zusammenfassung

Photonische Kristalle sind optisch aktive Materialien welche die Propagation von Licht innerhalb bestimmter Frequenzbereiche verhindern. An diesen Materialien wurden viele neue, hochinteressante optische Effekte beobachtet die beträchtlichen technologischen Fortschritten Vorschub geleistet haben. Die Erforschung des Designs der periodischen Strukturen von photonischen Kristallen ist einer der essentiellen Forschungszweige die sich mit der Optimierung von optischen Eigenschaften für technische Anwendungen befassen. In der Natur haben viele lebende Organismen biologische photonische Kristalle evolviert die in der Lage sind bunte, irisierende Farben hervorzubringen. Diese biologischen Modellsysteme, besonders die photonischen Strukturen welche von der Kutikula der Insekten gebildet werden, haben sich bereits als eine ergiebige Inspirationsquelle für Design und Entwicklung von synthetischen optischen Materialien erwiesen.

Im Rahmen dieser Arbeit habe ich die Beziehungen von Struktur und optischen Eigenschaften der kutikulären photonischen Kristalle von fünf Käferarten aus drei verschiedenen taxonomischen Gruppen untersucht um deren Designprinzipien aufzuklären und zu verstehen. Mikrostruktur und Zusammensetzung dieser photonischen Kristalle wurden unter Anwendung eines breiten Spektrums von Methoden aus der Mikroskopie und chemischen Analyse experimentell charakterisiert. Ihre optischen Eigenschaften wurden sowohl experimentell gemessen als auch anhand von auf der Strukturanalyse basierenden Modellen theoretisch charakterisiert.

Die Ergebnisse zeigen, dass die untersuchten Käferarten Mehrfachschichten, sogenannte Multilayer, und helikoidale Strukturen in unterschiedlichen Schichten ihrer Kutikula evolviert haben: erstere in der Epikutikula bei Laufkäfern und letztere in der Exokutikula bei Blatthornkäfern. Der Rüsselkäfer besitzt dreidimensionale, auf der bikontinuierlichen kubischen D- oder Diamantstruktur basierende photonische Kristalle die in Schüppchen gebildet werden welche Auswüchse der Kutikula darstellen. Durch Modifikationen von Struktur und Zusammensetzung dieser Grundstrukturen erreichen die untersuchten Käferarten optische Eigenschaften die für die jeweils wirkenden ökophysiologischen Belastungen optimiert sind. In einigen Fällen wurden dabei ungewöhnliche und unerwartete optische Effekte beobachtet. Die

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Zusammenfassung

Prinzipien sowohl der grundlegenden Architektur, als auch der zusätzlichen Modifikationen bieten Inspiration für das Design synthetischer Komposit- Materialien die nicht nur neuartige optische Eigenschaften bieten sondern diese mit anderen physikalischen Eigenschaften in einem ausgeglichenen Gleichgewicht kombinieren.

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